Plant Molecular Biology Reporter 16: 918, 1998. 1998 Kluwer Academic Publishers. Printed in Belgium.

Commentary

Rapid High Quality RNA Preparation from Pine Seedlings

MANUEL G. CLAROS1 and FRANCISCO M. CNOVAS
Laboratorio de Bioqumica y Biologa Molecular, Facultad de Ciencias-Instituto Andaluz de Biotecnologa, Universidad de Mlaga, Mlaga, Spain

Abstract. Conventional RNA extraction methods have been shown to produce poor quality RNA when applied to pine and other gymnosperms. We present a protocol for extracting highly pure RNA from pine. Modications to conventional procedures include: 1) the use of seedlings, 2) the use of phenol and PVP to rapidly remove DNA, proteins and pigments, and 3) the use of salt precipitation to remove other contaminating compounds. The procedure can be completed in less than eight hours. Yield and purity were monitored by denaturing gel electrophoresis and by UV absorbance (A 260 /A280 and A260 /A230 ). These ratios were over 2, indicating an absence of contaminating metabolites. Additionally, a new absorbance ratio (A260 /A210 ) was introduced to monitor the RNA purity in each step (it indicates the ratio of covalent links in the solution belonging to RNA). The yield was around 300 g total RNA per gram of tissue of Pinus sylvestris and over 400 g of total RNA per gram of P. pinaster tissue, which is a high recovery (more than 63%) for gymnosperms. The RNA was of sufcient quality for use in a RT-PCR reaction that amplied 1 kb of the pine GS gene. This protocol has been applied with success to other woody plants like Populus species. Abbreviations: DEPC, diethyl pyrocarbonate; AMV, avian myeloblastosis virus; FW, fresh weight. Key words: RNA extraction, pine, seedling

Introduction Protocols for RNA extraction (Chirgwin et al., 1979; Hughes and Galau, 1988) have been successfully applied to many plant species. These protocols have been further modied to obtain RNA from differentiated tissues and organs and they can provide RNA suitable for several kinds of analyses (Cantn et al., 1993; Chang et al., 1993; Chinn and Silverthorne, 1993; Bugos et al., 1995). The use of CsCl gradients (Yamamoto et al., 1991; Lewinsohn et al., 1994), several additional precipitation steps (Forreiter and Apel,
1 Author for correspondence: e-mail: claros@uma.es; fax: 34 5 213 20 00.

10 1993; Arahira and Fukazawa, 1994), and/or anion-exchange chromatography (Chinn and Silverthorne, 1993) will produce high quality RNA. However, in most cases, gymnosperm RNA puried by these methods can only be used for blotting analyses, or the yield is too low for most applications. One pine species, Pinus taeda, does respond to such methods (Zhang and Chiang, 1997). Others, like P. sylvestris or P. pinaster, yield RNA only suitable for blot analyses (Cantn et al., 1993; Garca-Gutirrez et al., 1995) due to the presence of polysaccharides, secondary metabolites and pigments that co-purify with RNA and inhibit the enzyme modications required for downstream applications (Pandey et al., 1996; M.G. Claros, A. Garca-Gutirrez and F. M. Cnovas, unpublished results). We now present a modied protocol that can produce RNA of high yield and purity in less than eight hours from pine seedlings.

Materials and Methods Plant Materials P. sylvestris and P. pinaster seeds were supplied by Instituto Nacional para la Conservacin de la Naturaleza (Madrid, Spain), imbibed for 24 hours in water and germinated in moistened vermiculite at 22 C for 912 days under a 16 h day. Solutions The following solutions were prepared from Milli-Q autoclaved water and powder stocks, or from Milli-Q autoclaved stocks (Huang et al., 1995). DEPC treatment of the water was not required. TS: 0.1M Tris-HCl pH 7.4; 1% SDS; and 5% 2-mercapto-ethanol. Dissolve and autoclave all but the 2-mercapto-ethanol which should be added prior to use. TE: 10 mM Tris-HCl pH 7.4; 1 mM EDTA. Autoclaved. Liquid nitrogen. Mortar and pestle treated 2 h at 180 C in an oven. AP: acid phenol (pH 4.3, Amresco). Store at 4 C. Chloroform (Panreac). Insoluble PVP (Sigma P-6755). Na-acetate: 4M autoclaved (pH not adjusted). NH4 -acetate: 3M autoclaved (pH not adjusted). LiCl: 8M autoclaved and stored at 4 C. EDTA: 0.2M autoclaved.

11 Ethanol: 100% and 70% stored at room temperature. 2-Propanol: stored at room temperature. CTAB/NaCl: 10% N-cetyl-N,N,N-trimethylammonium bromide (Paureac); and 1,4 M NaCl.

Protocol RNA Extraction 1. Harvest seedlings when their cotyledons are 1.52 cm (just before the cotyledons leave the megagametophyte). 2. Cool the mortar and pestle and grind 6 g of seedlings1 to a ne powder in the presence of liquid nitrogen. Do not let the tissue thaw. 3. Transfer the powder to a prechilled polypropylene tube and add 3 mL of TS, 2 mL of AP and 1 mL chloroform. Stir and vortex until a complete emulsion is formed. 4. Spin for 5 min at 5500g and 4 C. 2 Transfer the top aqueous phase3 to a new prechilled polypropylene tube. 5. Extract the bottom green organic phase with 2 mL of TS and centrifuge as above. (The top phase will be green, add it to the previous aqueous phase) 6. Add 1 volume of AP:chloroform 1:1, mix vigorously until a complete emulsion forms and centrifuge for 5 min at 5500 g at 4 C until complete. 7. Extract with one volume of chloroform and centrifuge like before. 8. Add 600 mg of PVP and mix thoroughly. Incubate at 7580 C for 20 min to remove phenolic compounds. Mix gently from time to time.4 9. Cool at room temperature. Add 1 volume of water5 and 1 volume of chloroform. Mix to form an emulsion. 10. Spin at 5500g for 5 min. Take the top aqueous phase.6 11. Add 2 mL of CTAB/NaCl and mix well. Add 15 mL of chloroform and mix until complete emulsion. 12. Centrifuge at 5500g for 10 min at 4 C and collect supernatant. 13. Add 0.08 volumes of 4M Na-acetate to bring the solution to a concentration of 0.3 M Na-acetate, and mix well.7 14. Add 2 volumes of ethanol, hold at 4 C for 10 min.8 15. Centrifuge at 4 C for 10 min at 8500g. Discard the supernatant. 16. Rinse the pellet with 5 mL of 70% ethanol and dry it by vacuum. 17. Dissolve it in 3 mL TE by vortexing and pipeting up and down. 18. Centrifuge at 4 C for 5 min at 8500g to clean the aqueous solution since it will be cloudy. Discard the pellet and take the supernatant.

12 19. Determine RNA concentration and purity at this stage to determine if the extraction is correct and it is worthwhile to continue. 20. Add 0.1 volume of 3M NH4 -acetate and 0.8 volume of 2-propanol to the aqueous phase, and mix gently. Precipitate 15 min at 4 C. The RNA will form a cloud. 21. Centrifuge for 15 min at 8500g and 4 C. Pour off the supernatant and rinse with 5 mL of 70% ethanol at room temperature. 22. Spin 5 min at 8500g and dry the pellet at room temperature by inverting tubes on a paper towel or by vacuum. 23. Redissolve the pellet in 2 mL of TE, add 1 mL of 0.2 M EDTA and 1 mL of 8 M LiCl to bring the solution to a nal concentration of 50 mM EDTA and 2M LiCl. 24. Precipitate 1 h at 4 C (longer precipitations are not necessary) and collect the precipitate by centrifugation for 15 min at 8500g, 4 C. 25. Pour off the supernatant and rinse the pellet with 70% ethanol. Let the pellet dry for a few seconds under a low vacuum. 26. Dissolve the RNA pellet in 1 mL of TE. Calculate the concentration with 5 or 10 L. 27. Add 2 mL of ethanol to store at 20 C. When it is needed for use, add 1 volume of 3 M NH4 -acetate or 3 M Na acetate to precipitate the RNA. 30
Notes The protocol can be scaled up and down by appropriate changes in the quantities used. The centrifugation speeds and times given throughout the protocol can be increased but decreasing either of them is not recommended. Use swinging bucket rotors. 3. This phase should turn red due to the acidic pH. The volume recovered in mL is usually similar to the fresh weight (FW) in g. 4. The solution will turn green-yellow. During incubation, gentle mixing is recommended from time to time until all of the powder is in the bottom of the tube. 5. Water is added to increase recovery. 6. Be careful to take only the top phase and not the white band at the interface. A second chloroform extraction should be performed if the aqueous phase is cloudy due to the presence of PVP bound to phenols or polysaccharides. 7. If insoluble particles appear, spin at 8500g for 10 min at 4 C to remove them. Carefully take the supernatant since the pellet may not be compact. 8. RNA precipitations using alcohols are carried out at 4 C, at this temperature the polysaccharides and salts are more soluble than RNA and will not precipitate (Arahira and Fukazawa, 1994). There is no need to decrease the temperature to 20 C to obtain good yields (Zeugin and Hartley, 1985). 1. 2.

Selection of poly(A)+ RNA The poly(A)+ RNA fraction was selected using oligo-dT (15) cellulose (Boehringer) following the protocol described by Sambrook (Sambrook et al., 1989).

13 This was followed by precipitation of the RNA with 0.2M NaCl and 2 volumes of ethanol to concentrate the solution and avoid coprecipitation of SDS. The RNA pellet should be rinsed with 70% ethanol to remove Cl . This anion can inhibit several modifying enzymes like reverse transcriptase (Sambrook et al., 1989). RT-PCR This protocol is based on the cDNA Synthesis Kit of Boehringer, but was modied to perform a PCR reaction after cDNA synthesis. The solutions are provided by the kit, and the PCR10X buffer with Taq polymerase was also obtained from Boehringer. Dissolve 2.5 g of total RNA or 200 ng of poly(A)+ RNA in 9 L sterilised distilled water. Add 2 L of oligo-dT(15) primer (0.8 gL1 ). For a nested RT-PCR add 2 L of a 10 M oligonucleotide. Denature secondary structures by incubation at 70 C for 10 min and quick chill on ice. Collect the contents of the tube by brief centrifugation. Add: 4 L of 5X synthesis buffer; 1 L of RNase inhibitor (25 UL1 ); 2 L of 10 mM dNTP mix; and 2 L of AMV reverse transcriptase (20 UL1 ). Incubate the reaction for 10 min at room temperature, and for 60 min at 42 C. Terminate the reaction by incubating the tube at 90 C for 5 min and place on ice for 10 min.1 Collect the reaction by brief centrifugation and add 1 L of RNAse H. Incubate for 20 min at 37 C. Add 8 L of 10X PCR buffer; 5 L of each primer from a 10 M stock; 2L of 25 mM dNTP mix; 60 L of sterilised distilled water; and 1 L of Taq DNA polymerase (5 UL1 ). Mix gently, cover the reaction mixture with mineral oil (if necessary) and perform PCR. PCR cycle: denaturation was carried out for 1 min at 91 C2 ; annealing for 1 min at 50 C; and extension for 2 min at 72 C; the cycle was repeated 30 times.
Notes 1. 2. If not denatured, AMV reverse transcriptase may interfere with subsequent amplications. Higher temperatures will affect the enzyme.

14 Results and Discussion We have devised a new RNA extraction method that eliminates the common interferences (sample oxidation, degradation, and secondary metabolite contamination) that prevented the isolation of high quality RNA from pine using conventional methods. The protocol can be accomplished in less than eight hours and is relatively inexpensive. Seedlings should be used fresh, although they are also suitable if frozen at 80 C for several weeks (longer periods like 612 months will diminish the RNA yield to nothing). Older stages accumulate contaminating substances and the RNA is harder to extract. Embryos or very young seedlings can be used to decrease the secondary metabolite contamination, although the mRNA expressed in these stages could not be representative. The buffer, TS, contains SDS to facilitate extraction and 2-mercapto-ethanol to prevent sample oxidation and to inhibit the RNAses released from the tissues prior to phenol extraction. There is no need to add isoamyl alcohol to chloroform in these extractions. Acid phenol (pH 4.4) is used to promote cell disruption and removal of DNA. The acidic pH helps to remove phenolic compounds after subsequent treatment with polyvinylpyrrolidone (PVP). PVP is a solid polymer that has a high molecular weight, is chemically inert, and forms a complex with polyphenols through hydrogen bonding, allowing them to be separated from RNA (thus the separation is improved by lowering the pH). PVP is more efcient than a similar polymer, polyvinyl polypyrrolidone (PVPP), used in other protocols (Porebski et al., 1997). Insoluble PVP instead of soluble PVP is preferred since soluble PVP will obstruct RNA precipitation. The rapid removal of phenolic compounds is done to inhibit the oxidation reactions which cause irreversable damage to RNA. The CTAB extraction removes most of copuried polysaccharides. After step 19, the A260/A230 ratio is 2.0, suggesting high purity of RNA (low polysaccharide or phenol contamination). However, the A260/A280 ratio is abnormally high, indicating that both ratios are not sufcient to monitor RNA purity. To better assess the level of purity, we have introduced the ratio A260/A210 to reect the amount of RNA in respect to other compounds in the same solution. Increases in this ratio indicate that the absorbance of covalent links is essentially due to RNA and not to other contaminating substances. The low A260/A210 ratio clearly indicates the presence of compounds other than RNA at stage 19 of the protocol. Furthermore, the occurrence of a smear on denaturing agarose gels suggests that RNA-phenol-polysaccharide complexes could be present (Ainsworth, 1994) and could disturb RNA migration (data not shown). Polysaccharides can also be removed by use of 2-butoxyethanol (Fluka) (Schultz et al., 1994). In our hands, this method does not

15
Table 1. Purity and yield ratios (including the standard deviation) for several protocol steps from at least three experiments and starting with different seedling amounts. Protocol step A260 /A280 19 22 26 A260 /A230 A260 /A210 g RNA/g FW % recovery 100 75.4 63.9

2.23 0.06 2.09 0.14 0.54 0.10 496 49 2.12 0.02 2.19 0.05 0.74 0.01 374 128 2.10 0.05 2.35 0.04 0.81 0.05 317 95

improve the RNA purity and the yield diminishes to half (around 150 g RNA per gram of fresh weight at the end of the process). Precipitation with NH4 -acetate removes traces of proteins, free nucleotides, and small nucleic acids (Crouse and Amorese, 1987), and increases the A260 /A210 and A260 /A230 ratios while decreasing the A260 /A280 ratios to desirable values (Wilnger et al., 1997). However the yield is highly variable (see the standard deviation in step 22, Table 1). Usually, P. pinaster gives the best yields at this stage. The short precipitation with LiCl (in the presence of EDTA) removes DNA, tRNA and small nuclear RNA. It should be noted that the RNA precipitated with LiCl is a white precipitate. In previous descriptions, the RNA is brown after this step due to sample oxidation (Cantn et al., 1993; Lewinsohn et al., 1994). When pellets are colored after step 19 or 22, the sample only worked for blotting analyses. The three absorbance ratios at the end of the process (Table 1, step 26) indicate a lack of contamination from polysaccharides and polyphenols. The RNA yields are over 300 g per gram of fresh weight with P. sylvestris and 400 g/g FW with P. pinaster, and these are independent of the initial fresh weight (FW). By contrast, other woody plant RNA protocols do not yield 250 g RNA/g FW (Cantn et al., 1993; Lewinsohn et al., 1994; Bugos et al., 1995). The percentage of recovery is increased to over 63% of the initial RNA. Furthemore, the RNA can be stored in 66% ethanol for long periods at 20 C, and can undergo several cycles of freezing and thawing without nicking. This is due to the absence of metabolite contamination that makes RNA unstable (Lodhi et al., 1994). This offers an advantage since P. sylvestris seedlings stored for three weeks at 20 C yield less than 200 g per g of FW. The RNA that gave smeared bands in denaturing agarose gels after stage 22 provides discrete rRNA bands that have no apparent degradation after stage 26 (data not shown). The same protocol has been assayed in Populus hybrids and similar yields and purity were obtained (F. Gallardo, personal communication). This suggests that the protocol may be applied to other woody species.

16

Figure 1. Agarose electrophoresis of the RT-PCR reaction for both P. sylvestris total and poly(A)+ RNA. Each lane contains a 0.1 volume of the RT-PCR reaction that amplied the GS gene.

The clearest evidence of the quality and purity of the RNA is provided by an RT-PCR conducted on total and poly(A)+ RNA. The upstream primer used was 5 -GACAGAGAAGGTCATTG-3 , and the downstream primer was 5 CGTCTCAGCAATCAT-3 (from the P. sylvestris glutamine synthetase gene, Cantn et al., 1993). They amplied a 1056 bp fragment (Figure 1) as a discrete band when both the total RNA and poly(A)+ fraction were used as a

17 template. The absence of a smear demonstrates the lack of signicant RNA degradation.

Acknowledgements We thank Dr F. Gallardo and Dr C. vila for helpful discussions, and R. Crespillo for technical assistance. This work was supported by a Grant from Direccin General de Investigacin Cientca y Tcnica (PB95-0482), Junta de Andaluca (Research group CVI-0114) and Project of Technology Priority form the European Union (PTP31). MGC is a recipient of a Contrato de Reincorporacin de Doctores y Tecnlogos from the Ministerio de Educacin y Ciencia (Spain).

References
Ainsworth C (1994) Isolation of RNA from oral tissue of Rumex acetosa (Sorrel). Plant Mol Biol Rep 13: 198203. Arahira M and Fukazawa C (1994) Ginkgo 11S seed storage protein family mRNA: unusual Asn-Asn linkage as post-translational cleavage site. Plant Mol Biol 25: 597605. Bugos RC, Chiang VL, Zhang X-H, Campbell ER, Podila GK and Campbell WH (1995) RNA isolation from plant tissues recalcitrant to extraction in guanidine. BioTechniques 19: 734737. Cantn FR, Garca-Gutirrez , Gallardo F, de Vicente A and Cnovas FM (1993) Molecular characterization of a cDNA clone encoding glutamine synthetase from a gymnosperm, Pinus sylvestris. Plant Mol Biol 22: 819828. Chang S, Puryear J and Cairney J (1993) A simple and efcient method for isolating RNA from pine trees. Plant Mol Biol Rep 11: 113116. Chinn E and Silverthorne J (1993) Light-dependent chloroplast development and expression of a light-harvesting chlorophyll a/b binding protein gene in the gymnosperm Ginkgo biloba. Plant Physiol 103: 727732. Chirgwin JM, Przybyla AE, Macdonald RJ and Ratter WJ (1979) Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry 18: 5294 5299. Crouse J and Amorese D (1987) Ethanol precipitation: ammonium acetate as alternative to sodium acetate. Focus 9: 35. Forreiter C and Apel K (1993) Light-independent and light-dependent protochlorophyllide-reducing activities and two distinct NADPH-protochlorophyllide oxidoreductase polypeptides in mountain pine (Pinus mugo). Planta 190: 536545. Garca-Gutirrez , Cantn FR, Gallardo F, Snchez-Jimnez F and Cnovas FM (1995) Expression of ferredoxin-dependent glutamate synthase in dark-grown pine seedlings. Plant Mol Biol 27: 115128. Huang YH, Leblanc P, Apostolou B, Stewart B and Moreland RB (1995) Comparison of Milli-Q PF Plus water to DEPC-treated water in the preparation and analysis or RNA. BioTechniques 19: 656661.

18
Hughes DW and Galau G (1988) Preparation of RNA from cotton leaves and pollen. Plant Mol Biol Rep 6: 253257. Lewinsohn E, Steele CL and Croteau R (1994) Simple isolation of functional RNA from woody stems of Gymnosperms. Plant Mol Biol Rep 12: 2025. Lodhi MA, Ye G-N, Weeden NF and Reisch BI (1994) A simple and efcient method for DNA extractions from grapevine cultivars and Vitis species. Plant Mol Biol Rep 12: 613. Pandey, RN, Adams RP and Flournoy LE (1996) Inhibition of random amplied polymorc DNAs (RAPDs) by plant polysaccharides. Plant Mol Biol Rep 14: 1722. Porebski S, Bailey LG and Baum BR (1997) Modication of a CTAB DNA extraction protocol for plants containing high polysaccharide and polyphenol components. Plant Mol Biol Rep 15: 815. Sambrook KJ, Fritsch EF and Maniatis T (1989) Molecular Cloning. Cold Spring Harbour Laboratory Press, Cold Spring Harbour. New York. Schultz DJ, Craig R, Cox-Foster DL, Mumma RO and Medford JI (1994) RNA isolation from recalcitrant plant tissue. Plant Mol Biol Rep 12: 310316. Wilnger WW, Mackey K and Chomczynski P (1997) Effect of pH and ionic strength on the spectrophotometric assessment of nucleic acid purity. BioTechniques 22: 474481. Yamamoto N, Mukai Y, Matsuoka M, Kano-Murakami Y, Tanaka Y, Ohashi Y, Ozeki Y and Odani K (1991) Light-independent expression of cab and rbcS genes in dark-grown pine seedlings. Plant Physiol 95: 379383. Zeugin JA and Hartley JL (1985) Ethanol precipitation of DNA. Focus 7: 12. Zhang S-H and Chiang JL (1997) Molecular cloning of 4-coumarate coenzyme A ligase in loblolly pine and the roles of this enzyme in the biosynthesis of lignin in compression wood. Plant Physiol 113: 6575.