- Email: [email protected]
Isolation of high-quality nucleic acids from Cistus creticus ssp. creticus and other medicinal plants
ANALYTICAL BIOCHEMISTRY Analytical Biochemistry 328 (2004) 90–92 www.elsevier.com/locate/yabio
Notes & Tips
Isolation of high-quality nucleic acids from Cistus creticus ssp. creticus and other medicinal plants夽 Irene Pateraki and Angelos K. Kanellis¤ Group of Biotechnology of Pharmaceutical Plants, Laboratory of Pharmacognosy, Department of Pharmaceutical Sciences, Aristotle University of Thessaloniki, 541 24 Thessaloniki, Greece Received 11 November 2003
EDTA, 1.5% (w/v) sodium dodecyl sulfate, 1% (w/v) sodium deoxycholate, 1% (v/v) Igepal CA 630 (Fluka Chemie, Gmbh., Buchs, Switzerland), 5 mM thiouria, 10 mM dithiothreitol, 1 mM aurintricarboxylic acid]. Add 6 ml of 20% polyvinylpyrrolidone (PVP)1 just before homogenization. Homogenize the solution with Polytron PT 2100 (Kinematica, Switzerland) at full speed for approx. 30 s. Centrifuge the homogenate at 10,000g for 15 min at 4 °C to remove insoluble particulates. Filter the supernatant through sterilized miracloth (Calbiochem, La Jolla, CA, USA) into an Oak Ridgetype tube. Precipitate nucleic acids with 1 volume isopropanol and 0.1 volume of 3 M sodium acetate. Incubate at ¡20 °C for at least 1 h. Centrifuge the solution at 10,000g for 20 min and resuspend the pellet in 6 ml extraction buVer. Extract the sample with equal volume of nonequilibrated phenol/chloroform. Centrifuge the mixture at 10,000g for 10 min at 4 °C and rescue the upper phase. Add CTAB and NaCl at Wnal concentrations of 1% (w/v) and 0.7 M, respectively. Incubate the mixture at 65 °C for 15 min and extract the solution with equal volume of chloroform, for carbohydrate removal. Centrifuge the mixture at 10,000g for 10 min at 4 °C and rescue the upper aqueous phase. Add 10 M LiCl to a Wnal concentration of 3 M for selective RNA precipitation. After at least 2 h incubation at ¡20 °C centrifuge the sample at 10,000g for 30 min. Resuspend the pellet in 500 l diethyl pyrocarbonate (DEPC)-treated water and perform a Wnal precipitation
Leaves of Cistus creticus ssp. creticus (Cistaceae) secrete several labdane-type diterpenes, which display either cytotoxic activity against a number of human leukemic cell lines or antibacterial and antifungal activity [1,2]. Considering the characteristics and possible future applications of these metabolites it seemed worthwhile to study their biosynthesis and metabolism at the molecular level. Thus, it was essential to establish methods for nucleic acid extraction and puriWcation from C. creticus tissues. Cistus species contain high amounts of metabolites that interfer with nucleic acid isolation, such as terpenoids, polyphenols, Xavonoids, Xavonoid aglycones, and glycosides, resin, and thick waxy cuticle covering the aerial parts’ epidermis and especially the leaves [3,4]. As a consequence of these characteristics, several published methods or kits for nucleic acid isolation (e.g., Nucleospin RNA plant kit; Macherey–Nagel, Duren, Germany) [5–10] that were applied to this plant had unsatisfactory results. The only method that gave good-quality total RNA was the one described in Loulakakis et al. [8] but the yield was relatively low (data not shown). In this report, we describe optimized protocols that yield large amounts of highquality genomic DNA and total RNA from C. creticus tissues. The described methods were appropriate for nucleic acid isolation independently of the sampling period, plant age, or plant cultivation method and worked eYciently for other medicinal plant species also. The total RNA extraction procedure is as follows: I Grind 1 g of leaf tissue in Wne powder with mortarl and pestle in liquid nitrogen and place it in a sterile 50-ml tube with 20 ml prechilled extraction buVer [200 mM Tris–HCl, pH 8.5, 300 mM LiCl, 10 mM
I
夽 This research was supported by a grant from the Greek General Secretariat of Research and Technology (PENED 99ED 637) awarded to A.K.K. ¤ Corresponding author. Fax: +30-2310-997662. E-mail address: [email protected] (A.K. Kanellis).
1 Abbreviations used: PVP, polyvinylpyorolidone; DEPC, diethyl pyrocarbonate; FW, fresh weight; CTAB, cetyltrimethylammonium bromide.
0003-2697/$ - see front matter 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2004.01.030
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Notes & Tips / Analytical Biochemistry 328 (2004) 90–92
step with 2 volumes of absolute ethanol and 0.1 volume of 3 M NaAC, pH 5.2, with at least 2 h incubation at ¡20 °C. I Spin down the RNA solution for 30 min at 4 °C, resuspend the pellet in 100 l DEPC-treated water, and store at ¡80 °C. For genomic DNA extraction a similar procedure with slight diVerences was followed. The RNA extraction buVer was used in a diVerent ratio [10 ml buVer per gram of fresh weight (FW) tissue with 3 ml 20% PVP] without aurintricarboxylic acid. Cell disruption was achieved using heat treatment (65 °C for 1–2 h), because mechanical cell disruption (homogenization) resulted in slightly sheared DNA (data not shown). Heat-treated samples yielded approximately 10 times more DNA than non-heat-treated samples (data not shown). Isopropanol precipitation was performed at room temperature to reduce the carryover salts and the pellet was resuspended in 5 ml TE buVer (10 mM Tris–HCl, pH 8.0, and 1 mM EDTA, pH 8.0) prior to the phenol/chloroform step. Tris–HCl pH 8.0, equilibrated phenol was used for DNA isolation instead of nonequilibrated phenol, and the LiCl step was replaced with a second isopropanol precipitation for eYcient DNA isolation. All glass- and plasticware were autoclaved for 30 min at 125 °C prior to use, while solutions for RNA extraction were treated with 0.1% DEPC for at least 3 h and then autoclaved; 20% PVP solution was prepared by dissolving the PVP K30 (MW 40,000) powder in DEPCtreated water. Nonequilibrated phenol/chloroform reagent was prepared by mixing 500 g of crystal phenol with 150 ml DEPC-treated water, 70 ml m-Cresol, and 0.5 g 8-hydroxyquinoline, supplemented with equal volume of chloroform. Tris–HCl, pH 8.0, equilibrated phenol was prepared according to Sambrook et al. [11]. A critical step for successful RNA preparation was the ratio of extraction buVer volume to C. creticus FW tissue. Ten milliliters buVer per gram of FW tissue supplemented with 3% PVP gave satisfactory results, with regard to total RNA quality and quantity, for growthchamber-cultivated plants. For Weld-grown plants, it was necessary to use 20 ml extraction buVer and 6% PVP for 1 g of FW tissue to obtain good-quality RNA. These diVerences are probably due to variation in polyphenol content in these samples [4]. Addition of PVP and not polyvinylpolypyrrolidone in the extraction buVer during the cell lysis procedure was important as this resulted in 25% higher nucleic acid yields (data not shown). Soluble PVP was subsequently removed from the solution during the chloroform extraction step. The detergents contained in the extraction buVer proved to be of great signiWcance, because no nucleic acids were isolated when extraction buVers with no detergents (or lower concentrations of them) were used (data not shown). They helped in more eYcient cell lysis (synergistically with heat treatment for DNA and
91
mechanical homogenization for RNA) as they dissociate cell membranes. Moreover they were probably responsible for the breakdown of complexes formed between nucleic acids and insoluble cell particles that are precipitated in the Wrst step of most protocols used during which cell debris were discarded. Another advantage is the omission of the ultracentrifugation step described in Loulakakis et al. [8]. Protein and polysaccharide removal were achieved by phenol/chloroform and CTAB/chloroform extractions, respectively. The presence of discrete ribosomal RNA bands (Fig. 1A) suggests that RNA degradation was negligible. Further, these RNA preparations were devoid of chromosomal DNA contamination. The method proved eYcient for total RNA extracted from Cistus creticus ssp. creticus, Myrtus communis, Lavandula hybrida, and Ebenus cretica leaves (Fig. 1A). Yield was satisfactory for all species, ranging from 118 to 194 g/g FW. The ratio A260/A280 was from 1.82 to 1.85, indicating the purity of the samples. Further, the quality of total RNA from C. creticus was tested by the successful application of RT-PCR (Fig. 1C) and RNA blot analysis (data not shown). The single sharp band, which was observed after RNA blot hybridization, indicated the absence of degradation (data not shown). For RT-PCR reactions cDNA was synthesized from C. creticus young leaves total RNA and oligo(dT) primer (50-ACTAGTCTCGA G(T)19-30). Synthesized cDNA was used as template for CcGGPPS1 and CcGGPPS2 (C. creticus geranylgeranyl diphosphate synthase gene 1 and 2, respectively) gene ampliWcation (Fig. 1C) using gene-speciWc primers
Fig. 1. Electrophoretic and RNA blot analysis of total RNA from diVerent plant species. (A) 5 g of total RNA extracted from Cistus creticus ssp. creticus (lane 1), Lavandula hybrida (lane 2), Myrtus communis (lane 3), and Ebenus cretica (lane 4), and from C. creticus roots (lane 5), Xower buds (lane 6), stems (lane 7), and leaves (lane 8) analyzed in 1% agarose gel stained with ethidium bromide. (B) Total RNA extracted from young leaves (lanes 1 and 3) and glandular trichomes (lanes 2 and 4) of C. creticus visualized in agarose gel with ethidium bromide (lanes 1 and 2), or blotted and hybridized with radiolabeled CcGGPPS1 (lanes 3 and 4). (C) RT-PCR products for CcGGPPS 30 ampliWcation (lane 1, molecular weight markers; lane 2, CcGGPPS1; lane 3, CcGGPPS2) resulted from cDNA synthesized from total RNA isolated from C. creticus young leaves.
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Notes & Tips / Analytical Biochemistry 328 (2004) 90–92
(GGPPS1.FOR 50-GTTGGCGTATCCCCCGCCCG-30 and GGPPS2.FOR 50-TGGGGGCCTCGCCGGAT AA-30) and oligo(dT). The protocol developed here was used to isolate total RNA from C. creticus glandular trichomes, roots, Xower buds, and stems with excellent results (Figs. 1A and B). Genomic DNA extraction from leaves of C. creticus ssp. creticus, Salvia oYcinalis, M. communis, L. hybrida, and E. cretica yielded 238, 127, 93, 173, and 14 g DNA per gram of tissue FW, respectively. All samples were intact with no shearing and exhibited good gel migration (Fig. 2A). The yield was estimated by measuring the absorbance at 260 nm after RNase treatment while the A260/A280 ratio, ranging from 1.81 § 0.09 to 1.94 § 0.06, indicated the purity of the preparations. DNA from all samples was eVectively subjected to complete digestions with HindIII restriction endonuclease (Fig. 2A) and to PCR ampliWcation (Fig. 2B). For PCRs, genomic DNA extracted from all plants mentioned above was used as template in diVerent concentrations, from 5 to 100 ng, with equal eYciency (data not shown), implying the quality and purity of the extracts. 18S ribosomal DNA was selected for ampliWcation, because it is highly conserved among all plant species, using the 18S.FOR 50-CTGGTTGATCCTGCCAGT-30 and 18S.REV 50ATTACCGCGGNTGCTGGC-30 primers. Genomic DNA from C. creticus digested by several restriction
Fig. 2. Electrophoretic and Southern blot analysis of genomic DNA samples. (A) Genomic DNA extracted from Cistus creticus ssp. creticus (lanes 2 and 8), Salvia oYcinalis (lanes 3 and 9), Lavandula hybrida (lanes 4 and 10), Myrtus communis (lanes 5 and 11), and Ebenus cretica (lanes 6 and 12) and analyzed in 1% agarose gel. In lanes 8–12 genomic DNA has been subjected to HindIII restriction endonuclease digestion. The 1-kb DNA ladder (Gibco BRL, Life Technologies) was used as molecular weight marker (lane 1). (B) PCR ampliWcation of a 560-bp 18S rDNA fragment using as template 25 ng genomic DNA extracted from C. creticus (lane 2), S. oYcinalis (lane 3), L. hybrida (lane 4), M. communis (lane 5), and E. cretica (lane 6). The 100-bp DNA ladder (Invitrogen) was used as molecular weight marker (lane 1). (C) Southern blot of C. creticus genomic DNA (10 g/lane) digested with EcoRI (lane 1), AvaI (lane 2), NdeI (lane 3), EcoRI–AvaI (lane 4), EcoRI–NdeI (lane 5), and AvaI–NdeI (lane 6) and hybridized with radiolabeled CcGGPPS1. Molecular weights (in bp) are indicated at the left.
endonucleases was used for Southern blot analysis of CcGGPPS1 gene, giving sharp and distinct hybridization signals (Fig. 2C). Consequently, the methods described here for total RNA and genomic DNA isolation can be used for extraction of superior-quality nucleic acids free of contaminants. They were applied eVectively to more than one plant species rich in secondary metabolites, independently of plant cultivation method or plant age. Nucleic acids isolated according to these methods were biologically active and were used successfully in several enzymatic reactions. Total RNA isolated was used eVectively for cDNA synthesis, RT-PCRs, and RNA blot analysis while genomic DNA could be ampliWed by PCR and completely digested with restriction endonucleases. Acknowledgment We thank Dr M. Kalamaki for reading the manuscript. References [1] I. Chinou, C. Demetzos, C. Harvala, C. Roussakis, J. Verbist, Cytotoxic and antibacterial labdane type diterpenes from the aerial parts of Cistus creticus, Planta Med. 60 (1994) 34–36. [2] C. Dimas, C. Demetzos, M. Marsellos, R. Sotiriadou, M. Malamas, D. Kokkinopoulos, Cytotoxic activity of labdane type diterpenes against human leukemic cell lines in vitro, Planta Med. 64 (1998) 208–211. [3] J.A. Bryant, DNA extraction, in: P.M. Dey, J.B. Harborne (Eds.), Methods in Plant Biochemistry, Academic Press, San Diego, 1997, pp. 1–12. [4] M. Stefanou, Y. Manetas, The eVects of seasons, exposure, enhanced UV-B radiation and water stress on leaf epicuticular and internal UV-B absorbing capacity of Cistus creticus: a Mediterranean Weld study, J. Exp. Bot. 48 (1997) 1977–1985. [5] U.M. Csaikl, H. Bastian, R. Brettschneider, S. Gauch, A. Meir, M. Schauerte, F. Scholz, C. Sperisen, B. Vorman, B. Ziegenhaagen, Comparative analysis of diVerent DNA extraction protocols: A fast, universal maxi-preparation of high quality plant DNA for genetic evaluation and phylogenetic studies, Plant Mol. Biol. Rep. 16 (1998) 69–86. [6] S. Poreski, L.G. Bailey, B.R. Baum, ModiWcation of a CTAB DNA extraction protocol for plants containing high polysaccharides and polyphenol components, Plant Mol. Biol. Rep. 15 (1997) 8–15. [7] R.Z. Fu, J. Wang, Y.R. Sun, P.C. Shaw, Extraction of genomic DNA suitable for PCR analysis from dried plant rhizomes/roots, BioTechniques 25 (1998) 796–798. [8] K.A. Loulakakis, K.A. Roubelakis-Angelakis, A.K. Kanellis, Isolation of high quantities of intact RNA from rich in phenolics and poor in RNA content grapevine tissues, Am. J. Enol. Viticult. 47 (1996) 181–185. [9] E. Lewinson, C.L. Steele, R. Croteau, Simple isolation of functional RNA from woody stems of gymnosperms, Plant Mol. Biol. Rep. 12 (1994) 20–25. [10] J. Logemann, J. Schell, L. Willmitzer, Improved method for the isolation of RNA from plant tissues, Anal. Biochem. 163 (1987) 16–20. [11] J. Sambrook, E.F. Fritsch, T. Maniatis, Molecular Cloning: A laboratory Manual, second ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1989.
Notes & Tips
Isolation of high-quality nucleic acids from Cistus creticus ssp. creticus and other medicinal plants夽 Irene Pateraki and Angelos K. Kanellis¤ Group of Biotechnology of Pharmaceutical Plants, Laboratory of Pharmacognosy, Department of Pharmaceutical Sciences, Aristotle University of Thessaloniki, 541 24 Thessaloniki, Greece Received 11 November 2003
EDTA, 1.5% (w/v) sodium dodecyl sulfate, 1% (w/v) sodium deoxycholate, 1% (v/v) Igepal CA 630 (Fluka Chemie, Gmbh., Buchs, Switzerland), 5 mM thiouria, 10 mM dithiothreitol, 1 mM aurintricarboxylic acid]. Add 6 ml of 20% polyvinylpyrrolidone (PVP)1 just before homogenization. Homogenize the solution with Polytron PT 2100 (Kinematica, Switzerland) at full speed for approx. 30 s. Centrifuge the homogenate at 10,000g for 15 min at 4 °C to remove insoluble particulates. Filter the supernatant through sterilized miracloth (Calbiochem, La Jolla, CA, USA) into an Oak Ridgetype tube. Precipitate nucleic acids with 1 volume isopropanol and 0.1 volume of 3 M sodium acetate. Incubate at ¡20 °C for at least 1 h. Centrifuge the solution at 10,000g for 20 min and resuspend the pellet in 6 ml extraction buVer. Extract the sample with equal volume of nonequilibrated phenol/chloroform. Centrifuge the mixture at 10,000g for 10 min at 4 °C and rescue the upper phase. Add CTAB and NaCl at Wnal concentrations of 1% (w/v) and 0.7 M, respectively. Incubate the mixture at 65 °C for 15 min and extract the solution with equal volume of chloroform, for carbohydrate removal. Centrifuge the mixture at 10,000g for 10 min at 4 °C and rescue the upper aqueous phase. Add 10 M LiCl to a Wnal concentration of 3 M for selective RNA precipitation. After at least 2 h incubation at ¡20 °C centrifuge the sample at 10,000g for 30 min. Resuspend the pellet in 500 l diethyl pyrocarbonate (DEPC)-treated water and perform a Wnal precipitation
Leaves of Cistus creticus ssp. creticus (Cistaceae) secrete several labdane-type diterpenes, which display either cytotoxic activity against a number of human leukemic cell lines or antibacterial and antifungal activity [1,2]. Considering the characteristics and possible future applications of these metabolites it seemed worthwhile to study their biosynthesis and metabolism at the molecular level. Thus, it was essential to establish methods for nucleic acid extraction and puriWcation from C. creticus tissues. Cistus species contain high amounts of metabolites that interfer with nucleic acid isolation, such as terpenoids, polyphenols, Xavonoids, Xavonoid aglycones, and glycosides, resin, and thick waxy cuticle covering the aerial parts’ epidermis and especially the leaves [3,4]. As a consequence of these characteristics, several published methods or kits for nucleic acid isolation (e.g., Nucleospin RNA plant kit; Macherey–Nagel, Duren, Germany) [5–10] that were applied to this plant had unsatisfactory results. The only method that gave good-quality total RNA was the one described in Loulakakis et al. [8] but the yield was relatively low (data not shown). In this report, we describe optimized protocols that yield large amounts of highquality genomic DNA and total RNA from C. creticus tissues. The described methods were appropriate for nucleic acid isolation independently of the sampling period, plant age, or plant cultivation method and worked eYciently for other medicinal plant species also. The total RNA extraction procedure is as follows: I Grind 1 g of leaf tissue in Wne powder with mortarl and pestle in liquid nitrogen and place it in a sterile 50-ml tube with 20 ml prechilled extraction buVer [200 mM Tris–HCl, pH 8.5, 300 mM LiCl, 10 mM
I
夽 This research was supported by a grant from the Greek General Secretariat of Research and Technology (PENED 99ED 637) awarded to A.K.K. ¤ Corresponding author. Fax: +30-2310-997662. E-mail address: [email protected] (A.K. Kanellis).
1 Abbreviations used: PVP, polyvinylpyorolidone; DEPC, diethyl pyrocarbonate; FW, fresh weight; CTAB, cetyltrimethylammonium bromide.
0003-2697/$ - see front matter 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2004.01.030
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Notes & Tips / Analytical Biochemistry 328 (2004) 90–92
step with 2 volumes of absolute ethanol and 0.1 volume of 3 M NaAC, pH 5.2, with at least 2 h incubation at ¡20 °C. I Spin down the RNA solution for 30 min at 4 °C, resuspend the pellet in 100 l DEPC-treated water, and store at ¡80 °C. For genomic DNA extraction a similar procedure with slight diVerences was followed. The RNA extraction buVer was used in a diVerent ratio [10 ml buVer per gram of fresh weight (FW) tissue with 3 ml 20% PVP] without aurintricarboxylic acid. Cell disruption was achieved using heat treatment (65 °C for 1–2 h), because mechanical cell disruption (homogenization) resulted in slightly sheared DNA (data not shown). Heat-treated samples yielded approximately 10 times more DNA than non-heat-treated samples (data not shown). Isopropanol precipitation was performed at room temperature to reduce the carryover salts and the pellet was resuspended in 5 ml TE buVer (10 mM Tris–HCl, pH 8.0, and 1 mM EDTA, pH 8.0) prior to the phenol/chloroform step. Tris–HCl pH 8.0, equilibrated phenol was used for DNA isolation instead of nonequilibrated phenol, and the LiCl step was replaced with a second isopropanol precipitation for eYcient DNA isolation. All glass- and plasticware were autoclaved for 30 min at 125 °C prior to use, while solutions for RNA extraction were treated with 0.1% DEPC for at least 3 h and then autoclaved; 20% PVP solution was prepared by dissolving the PVP K30 (MW 40,000) powder in DEPCtreated water. Nonequilibrated phenol/chloroform reagent was prepared by mixing 500 g of crystal phenol with 150 ml DEPC-treated water, 70 ml m-Cresol, and 0.5 g 8-hydroxyquinoline, supplemented with equal volume of chloroform. Tris–HCl, pH 8.0, equilibrated phenol was prepared according to Sambrook et al. [11]. A critical step for successful RNA preparation was the ratio of extraction buVer volume to C. creticus FW tissue. Ten milliliters buVer per gram of FW tissue supplemented with 3% PVP gave satisfactory results, with regard to total RNA quality and quantity, for growthchamber-cultivated plants. For Weld-grown plants, it was necessary to use 20 ml extraction buVer and 6% PVP for 1 g of FW tissue to obtain good-quality RNA. These diVerences are probably due to variation in polyphenol content in these samples [4]. Addition of PVP and not polyvinylpolypyrrolidone in the extraction buVer during the cell lysis procedure was important as this resulted in 25% higher nucleic acid yields (data not shown). Soluble PVP was subsequently removed from the solution during the chloroform extraction step. The detergents contained in the extraction buVer proved to be of great signiWcance, because no nucleic acids were isolated when extraction buVers with no detergents (or lower concentrations of them) were used (data not shown). They helped in more eYcient cell lysis (synergistically with heat treatment for DNA and
91
mechanical homogenization for RNA) as they dissociate cell membranes. Moreover they were probably responsible for the breakdown of complexes formed between nucleic acids and insoluble cell particles that are precipitated in the Wrst step of most protocols used during which cell debris were discarded. Another advantage is the omission of the ultracentrifugation step described in Loulakakis et al. [8]. Protein and polysaccharide removal were achieved by phenol/chloroform and CTAB/chloroform extractions, respectively. The presence of discrete ribosomal RNA bands (Fig. 1A) suggests that RNA degradation was negligible. Further, these RNA preparations were devoid of chromosomal DNA contamination. The method proved eYcient for total RNA extracted from Cistus creticus ssp. creticus, Myrtus communis, Lavandula hybrida, and Ebenus cretica leaves (Fig. 1A). Yield was satisfactory for all species, ranging from 118 to 194 g/g FW. The ratio A260/A280 was from 1.82 to 1.85, indicating the purity of the samples. Further, the quality of total RNA from C. creticus was tested by the successful application of RT-PCR (Fig. 1C) and RNA blot analysis (data not shown). The single sharp band, which was observed after RNA blot hybridization, indicated the absence of degradation (data not shown). For RT-PCR reactions cDNA was synthesized from C. creticus young leaves total RNA and oligo(dT) primer (50-ACTAGTCTCGA G(T)19-30). Synthesized cDNA was used as template for CcGGPPS1 and CcGGPPS2 (C. creticus geranylgeranyl diphosphate synthase gene 1 and 2, respectively) gene ampliWcation (Fig. 1C) using gene-speciWc primers
Fig. 1. Electrophoretic and RNA blot analysis of total RNA from diVerent plant species. (A) 5 g of total RNA extracted from Cistus creticus ssp. creticus (lane 1), Lavandula hybrida (lane 2), Myrtus communis (lane 3), and Ebenus cretica (lane 4), and from C. creticus roots (lane 5), Xower buds (lane 6), stems (lane 7), and leaves (lane 8) analyzed in 1% agarose gel stained with ethidium bromide. (B) Total RNA extracted from young leaves (lanes 1 and 3) and glandular trichomes (lanes 2 and 4) of C. creticus visualized in agarose gel with ethidium bromide (lanes 1 and 2), or blotted and hybridized with radiolabeled CcGGPPS1 (lanes 3 and 4). (C) RT-PCR products for CcGGPPS 30 ampliWcation (lane 1, molecular weight markers; lane 2, CcGGPPS1; lane 3, CcGGPPS2) resulted from cDNA synthesized from total RNA isolated from C. creticus young leaves.
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Notes & Tips / Analytical Biochemistry 328 (2004) 90–92
(GGPPS1.FOR 50-GTTGGCGTATCCCCCGCCCG-30 and GGPPS2.FOR 50-TGGGGGCCTCGCCGGAT AA-30) and oligo(dT). The protocol developed here was used to isolate total RNA from C. creticus glandular trichomes, roots, Xower buds, and stems with excellent results (Figs. 1A and B). Genomic DNA extraction from leaves of C. creticus ssp. creticus, Salvia oYcinalis, M. communis, L. hybrida, and E. cretica yielded 238, 127, 93, 173, and 14 g DNA per gram of tissue FW, respectively. All samples were intact with no shearing and exhibited good gel migration (Fig. 2A). The yield was estimated by measuring the absorbance at 260 nm after RNase treatment while the A260/A280 ratio, ranging from 1.81 § 0.09 to 1.94 § 0.06, indicated the purity of the preparations. DNA from all samples was eVectively subjected to complete digestions with HindIII restriction endonuclease (Fig. 2A) and to PCR ampliWcation (Fig. 2B). For PCRs, genomic DNA extracted from all plants mentioned above was used as template in diVerent concentrations, from 5 to 100 ng, with equal eYciency (data not shown), implying the quality and purity of the extracts. 18S ribosomal DNA was selected for ampliWcation, because it is highly conserved among all plant species, using the 18S.FOR 50-CTGGTTGATCCTGCCAGT-30 and 18S.REV 50ATTACCGCGGNTGCTGGC-30 primers. Genomic DNA from C. creticus digested by several restriction
Fig. 2. Electrophoretic and Southern blot analysis of genomic DNA samples. (A) Genomic DNA extracted from Cistus creticus ssp. creticus (lanes 2 and 8), Salvia oYcinalis (lanes 3 and 9), Lavandula hybrida (lanes 4 and 10), Myrtus communis (lanes 5 and 11), and Ebenus cretica (lanes 6 and 12) and analyzed in 1% agarose gel. In lanes 8–12 genomic DNA has been subjected to HindIII restriction endonuclease digestion. The 1-kb DNA ladder (Gibco BRL, Life Technologies) was used as molecular weight marker (lane 1). (B) PCR ampliWcation of a 560-bp 18S rDNA fragment using as template 25 ng genomic DNA extracted from C. creticus (lane 2), S. oYcinalis (lane 3), L. hybrida (lane 4), M. communis (lane 5), and E. cretica (lane 6). The 100-bp DNA ladder (Invitrogen) was used as molecular weight marker (lane 1). (C) Southern blot of C. creticus genomic DNA (10 g/lane) digested with EcoRI (lane 1), AvaI (lane 2), NdeI (lane 3), EcoRI–AvaI (lane 4), EcoRI–NdeI (lane 5), and AvaI–NdeI (lane 6) and hybridized with radiolabeled CcGGPPS1. Molecular weights (in bp) are indicated at the left.
endonucleases was used for Southern blot analysis of CcGGPPS1 gene, giving sharp and distinct hybridization signals (Fig. 2C). Consequently, the methods described here for total RNA and genomic DNA isolation can be used for extraction of superior-quality nucleic acids free of contaminants. They were applied eVectively to more than one plant species rich in secondary metabolites, independently of plant cultivation method or plant age. Nucleic acids isolated according to these methods were biologically active and were used successfully in several enzymatic reactions. Total RNA isolated was used eVectively for cDNA synthesis, RT-PCRs, and RNA blot analysis while genomic DNA could be ampliWed by PCR and completely digested with restriction endonucleases. Acknowledgment We thank Dr M. Kalamaki for reading the manuscript. References [1] I. Chinou, C. Demetzos, C. Harvala, C. Roussakis, J. Verbist, Cytotoxic and antibacterial labdane type diterpenes from the aerial parts of Cistus creticus, Planta Med. 60 (1994) 34–36. [2] C. Dimas, C. Demetzos, M. Marsellos, R. Sotiriadou, M. Malamas, D. Kokkinopoulos, Cytotoxic activity of labdane type diterpenes against human leukemic cell lines in vitro, Planta Med. 64 (1998) 208–211. [3] J.A. Bryant, DNA extraction, in: P.M. Dey, J.B. Harborne (Eds.), Methods in Plant Biochemistry, Academic Press, San Diego, 1997, pp. 1–12. [4] M. Stefanou, Y. Manetas, The eVects of seasons, exposure, enhanced UV-B radiation and water stress on leaf epicuticular and internal UV-B absorbing capacity of Cistus creticus: a Mediterranean Weld study, J. Exp. Bot. 48 (1997) 1977–1985. [5] U.M. Csaikl, H. Bastian, R. Brettschneider, S. Gauch, A. Meir, M. Schauerte, F. Scholz, C. Sperisen, B. Vorman, B. Ziegenhaagen, Comparative analysis of diVerent DNA extraction protocols: A fast, universal maxi-preparation of high quality plant DNA for genetic evaluation and phylogenetic studies, Plant Mol. Biol. Rep. 16 (1998) 69–86. [6] S. Poreski, L.G. Bailey, B.R. Baum, ModiWcation of a CTAB DNA extraction protocol for plants containing high polysaccharides and polyphenol components, Plant Mol. Biol. Rep. 15 (1997) 8–15. [7] R.Z. Fu, J. Wang, Y.R. Sun, P.C. Shaw, Extraction of genomic DNA suitable for PCR analysis from dried plant rhizomes/roots, BioTechniques 25 (1998) 796–798. [8] K.A. Loulakakis, K.A. Roubelakis-Angelakis, A.K. Kanellis, Isolation of high quantities of intact RNA from rich in phenolics and poor in RNA content grapevine tissues, Am. J. Enol. Viticult. 47 (1996) 181–185. [9] E. Lewinson, C.L. Steele, R. Croteau, Simple isolation of functional RNA from woody stems of gymnosperms, Plant Mol. Biol. Rep. 12 (1994) 20–25. [10] J. Logemann, J. Schell, L. Willmitzer, Improved method for the isolation of RNA from plant tissues, Anal. Biochem. 163 (1987) 16–20. [11] J. Sambrook, E.F. Fritsch, T. Maniatis, Molecular Cloning: A laboratory Manual, second ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1989.