RNA extraction from plant tissues

RNA Extraction From Plant Tissues RESEARCH 113 RNA Extraction From Plant Tissues The Use of Calcium to Precipitate Contaminating Pectic Sugars Valeriano Dal Cin,* Marcello Danesin, Fabio Massimo Rizzini, and Angelo Ramina Abstract Several protocols and commercial kits are used for the extraction of nucleic acids from different plant tissues. Although there are several procedures available to remove sugars, which hinder the extraction of clean genomic DNA, there are few to assist with extraction of RNA. Those presently used include precipitations with ethylene glycol monobutyl ether or lithium chloride (LiCl), or centrifugation in cesium chloride (CsCl) gradients, but these generally either do not allow high recovery of RNA, are time consuming, rely on hazardous chemicals or need special equipment. Here we present the use of the simple cation, Ca2+, which has been tested and shown to be very efficient for the precipitation of high molecular weight pectic sugars during RNA extraction. Results are presented for different plant tissues, especially tissues of peach and apple fruits at varying ripening stages. Index Entries: RNA extraction; RNA sugar removal; calcium; pectate; fruit. 1. Introduction Extraction of relatively pure nucleic acids is an important procedure and can be a limiting factor in molecular biology experiments. Plant tissues are characterized by a variable composition, and some tissues may be recalcitrant to RNA extraction. Purification of nucleic acids from plant tissues is often hindered by contaminating polysaccharides, polyphenolics, and secondary metabolites that, in the last steps of resuspension after precipitation by alcohol and salt, cause the formation of an opaque and viscous slurry that is difficult to separate from pelleted nucleic acids. The greater the sugar contamination, the greater is the difficulty in the subsequent use of RNA for Northern blotting, reverse transcription, electrophoresis, poly(A)+–RNA selection or quantification (1–4). Purification of RNA from sugar is generally achieved by precipitation with lithium chloride (LiCl) (5,6), salt–alcohol (7), ethylene glycol monobutyl ether (BE), or by cesium chloride (CsCl) centrifugation (2,3,7–9). These steps are time consuming, involve hazardous chemicals, and may increase RNA degradation. Despite these steps, in some cases, RNA quality and yield are poor. Although for DNA purification it is possible to use enzymes such as pectinase to remove some polysaccharides (10,11), this option is not feasible with RNA purification because of the risk of RNA degradation. In this article we propose the use of a cation, Ca2+, which may be added to samples at different stages of RNA extraction procedures, and that may make sugar precipitation possible while leaving nucleic acid in solution. We have tested calcium at different concentrations and applied as different compounds, using different parts of apple and peach fruits as test tissues. Calcium is likely to *Author to whom all correspondence and reprint requests should be addressed. University of Padua, Department of Environmental Agronomy and Crop Science, 35020, Agripolis, Legnaro, Italy. E-mail: [email protected]. Molecular Biotechnology © 2005 Humana Press Inc. All rights of any nature whatsoever reserved. 1073–6085/2005/31:2/113–120/$30.00 MOLECULAR BIOTECHNOLOGY 113 Volume 31, 2005 114 Dal Cin et al. work by precipitating pectin and its attached neutral sugar side chains, but efficiency may vary depending on the molecular weight and degree of esterification of the polyuronide chains. To our knowledge, this is the first example of using calcium for purification of nucleic acids from contaminating sugars. 2. Materials and Methods The peach variety used was Summer Rich, and the apple varieties were Golden Delicious, Royal Gala, and Stark Delicious. Plant material used for tests was: peach mesocarp (3 g) and epicarp (1.5 g), apple abscission zone (AZ) (0.5 g), peduncle (0.5 g), seed (0.5 g), cortex (3 g), and skin (1.5 g) at different ripening stages from a 12-mm diameter fruitlet to commercial harvest stage. Tissue was collected and immediately frozen in liquid nitrogen, then stored at –80°C. General RNA extraction procedures were as previously reported (12,13), with minor modifications, whereas for BE, the protocol used was as previously described (8,14). All solutions were prepared using water treated with diethyl pyro-carbonate (DEPC, 0.1% v/v) and autoclaved, and the term water refers to DEPC-treated water. Unless otherwise specified, solutions were used at 4°C. 2.1. Extraction With Calcium 1. Tissue was ground to a fine powder in liquid nitrogen using a mortar and a pestle. Ground powder was mixed with 10 mL of preheated (65°C) extraction buffer (100 mM Tris-HCl, pH 9.0, 100 mM NaCl, 5 mM EDTA, 2.5% [v/v] β-mercaptoethanol, 1% [w/v] SDS, 1% [w/v] PVP, 1% [w/v] PVPP, 5 mM ascorbic acid, 100 µg/mL proteinase K). Samples were incubated for 10 min at 65°C and briefly but vigorously shaken every 2–3 min. Samples should not be left for more than 10 min at 65°C, because extended times at 65°C can result in decreased purity of nucleic acids. 2. After incubation and immediately before centrifugation, calcium solution was added to samples. MOLECULAR BIOTECHNOLOGY 3. Samples were then briefly shaken vigorously, and cell debris pelleted at 10,000g at 4°C for 10 min. 4. The aqueous solution was then sequentially extracted in 10 mL of phenol preheated to 65°C, 10 mL of phenol–chloroform–isoamyl alcohol (25:24:1, by vol), and 10 mL of chloroform–isoamyl alcohol (24:1, v/v), each time shaking to mix thoroughly then centrifuging at 15,000g at 4°C for 10 min. 5. RNA was then precipitated by adding 1/10 vol of 3 M NaOAc, pH 4.8, and 1 vol of 2-propanol, and incubating at –80°C for 1 h. RNA was pelleted by centrifugation at 15,000g at 4°C for 30 min. Pellets were washed with 5 mL of 70% ethanol and air dried. 6. Pellets were resuspended in 600 µL of water and transferred to 1.5-mL microfuge tubes. To each sample was added 300 µL of 4X TBE, then 300 µL of 8 M LiCl. Solutions were gently mixed and allowed to stand at 4°C overnight. 7. Precipitated RNA was collected by centrifuging at 18,000g at 4°C for 30 min. Pellets were washed with 70% ethanol, vacuum dried, and resuspended in 100 µL of water. 2.2. Extraction With Ethylene GlycolBE Without Added Calcium To both assess and compare the effects of calcium, samples underwent either extraction without added calcium or a further purification with BE. In both cases step 2 was omitted and in the second case, samples underwent a further purification procedure after step 5, described as follows: 1. Pellets were resuspended in 10 mL (1 vol) resuspension buffer (25mM boric acid, 50 mM Tris-HCl, pH 7.6, 1.25 mM EDTA, pH 8.0, 0.1 M NaCl). 2. When dissolved, 4 mL (0.4 vol) of BE was added and tubes incubated in ice for 30 min. Carbohydrates were pelleted by centrifuging at 15,000g at 4°C for 10 min. 3. Supernatants were collected and 6 mL (0.6 vol) of BE added. Samples were incubated in ice Volume 31, 2005 RNA Extraction From Plant Tissues for 30 min and RNA collected by centrifuging at 15,000g at 4°C for 10 min. 4. Pellets were washed with 5 mL of 70% ethanol. Samples were then resuspended and precipitated with LiCl as previously described (steps 6 and 7 in Subheading 2.1.). 2.3. Calcium Compounds Tested and Other Calcium Treatments Calcium compounds used were calcium chloride and calcium hydroxide in water at a stock concentrations of 0.5 M. Calcium treatment was performed as in step 2 or after step 7 (in Subheading 2.1.) using 1 µL of calcium solution per 50 µL of resuspended pellet. In the latter case, calcium was added to samples, which were then vigorously shaken and immediately centrifuged at 18,000g for 5 min to pellet carbohydrates. RNA was then precipitated from the supernatants with 2-propanol and washed, as described in step 5 (Subheading 2.1.), vacuum dried, and resuspended. 2.4. Sample Evaluation All RNA extractions were repeated several times. The quality of the procedure was evaluated each time a pellet was visible. Diluted aliquots of samples were examined in a Perkin-Elmer spectrophotometer, and absorbance ratios A260/A230 and A260/A280 used as purity indexes. Total RNA concentration was calculated as follows: (A260 – A320) × (42.5) × dilution/(1000) = µg/µL. Samples (1µg of RNA) were separated on a 1% agarose gel in TAE buffer and stained with ethidium bromide. 2.5. Expression Studies Total RNA (30 µg) was treated with 10 units of RQ1 RNase-Free DNase (Promega, Milan, Italy) and 1 unit of RNAguard (RNase inhibitor) (Amersham Biosciences, Piscataway, NJ) for 30 min, then purified by extraction with phenol–chloroform (3:1), precipitated with 1 vol of 2-propanol and 1/10 vol of 3 M NaOAc at pH 4.8, incubated for 1 h at –80°C, centrifuged at 18,000g at 4°C for 30 min. Pellets were washed with 70% MOLECULAR BIOTECHNOLOGY 115 ethanol, vacuum dried, and resuspended in 50 µL of water. First-strand cDNA synthesis and semiquantitative polymerase chain reaction (PCR) to estimate the expression level of ubiquitin (MDU74358) using reverse transcriptase was performed as previously described (15). Ubiquitin-specific primers used were: (F = 5'-CATCCCCCCAGAC CAGCAGA-3'; R = 5'-ACCACGGAGACGCAA CACCAA-3'). The PCR amplification conditions were 10 min at 95°C (1 cycle), 94°C for 30 s, annealing at 62°C for 30 s, and extension at 72°C for 30 s (40 cycles) and 7 min at 72°C (1 cycle). The intensity of the band of this housekeeping gene on an ethidium-stained gel was used as an estimate of the expression level. Northern blotting was performed as previously described (14) and blots were hybridized with a radioactively labeled ubiquitin probe as previously described (14). 3. Results Because the apple fruitlet peduncle was the most difficult tissue as a result of the formation of large amounts of gel during extraction, the results presented regard especially this sample. The calcium effect was observable immediately after cell debris pelletting when a thick and gel-like layer was visible rather than a fluffy accumulation. In the absence of added calcium, the amount of water necessary for RNA resuspension was approx 10 times that necessary to hydrate the pellet from calcium-extracted RNA. The RNA purity was good for calcium-extracted RNA and BE-extracted RNA, but it was substantially worse for samples extracted without calcium (Table 1). Additional differences were observed when 1 µg of RNA was separated on an elecrophoresis gel, whereas RNA extracted without calcium migrated less distance than calcium-extracted RNA and with a “smile” typical of dirty RNA (Fig. 1A). RNA extracted with BE gave a pattern similar to calcium-extracted RNA (results not shown). The quantity of RNA extracted with added calcium was less than the amount obtained without calcium, although yield loss was only approx 5 to Volume 31, 2005 116 Dal Cin et al. Table 1 Comparison Between Parameters in Different RNA Extracting Procedures T0 Ca2+ Quantity (µg/g fw) A260/A230 A260/A280 Volume (µL) No Ca2+ Av. SD Av. SD 153 2.2 2 100 ±18 ±0.19 ±0.18 170 0.8 0.9 1500 ±20 ±0.12 ±0.14 BE Av. 12 3 1.8 10 Ca(OH)2 T20 CaCl2 SD Av. SD Av. SD Av. SD ±0.2 ±0.22 ±0.15 150 2.2 2 100 ±21 ±0.16 ±0.13 155 2.2 2 100 ±19 ±0.18 ±0.16 190 2.5 2.4 100 ±23 ±0.19 ±0.21 RNA was extracted with calcium (Ca2+), without calcium (No Ca) or with ethylene glycol monobutyl ether (BE). Sample was centrifuged immediately after calcium hydroxide (Ca(OH)2) or calcium chloride (CaCl2) addition (T0) or after 20 min from calcium addition (T20). The quantity indicates the amount of RNA in microgram extracted from a gram of sample fresh weight. A260/A230 and A260/A280 are the purity indexes for sugars and proteins and the volume is the amount of water in microliters used to hydrate RNA pellet. Av and SD indicates, respectively, the average and standard deviation of a least five experiments. Fig. 1. (A) RNA extracted without (NCa) and with calcium (Ca). (B) RNA from sample centrifuged after 20 min from calcium addition (20Ca) or immediately after calcium hydroxide (Ca(OH)2) or calcium chloride (CaCl2) addition. 10%. The amount of RNA obtained with the BE method was, however, quite low. Extraction in the presence of BE gave RNA that was relatively free of contamination by sugar (high A260/A230 ratio) and was comparable to calcium-extracted RNA in terms of protein content (A 260 /A 280 ratio; Table 1). From the results obtained, there was no difference between the two salts used as the source of Ca2+. Allowing calcium to act for as long as 20 min increased RNA purity, but some RNA degradation occurred (Fig. 1B and Table 1). The AZ and peduncle were the tissues most recalcitrant for RNA extraction, and in the case of the peduncle, the method with BE was ineffective at giving sufficient yield of RNA. The quantity of calcium to be added to give the best results in MOLECULAR BIOTECHNOLOGY terms of yield and quality (Table 2) depended on both the type of tissue and age (Table 3). Calcium at supraoptimal concentrations was detrimental to RNA quality because it increased degradation, giving a result similar to Fig.1B where calcium was left acting for more than 20 min. In addition, the optimal quantity of calcium to be applied varied depending on the season in which tissue was harvested (data not shown). Results presented were for a particularly dry and sunny year characterized by high sugar accumulation in the fruits. However, in a more typical season the effect of calcium was also beneficial but the optimal amount to be used was threefold less (data not shown). Calcium may be used during or before subsequent RNA purification steps (before or after LiCl precipitation or DNase treatment). From our research calcium was more effective when applied in diluted RNA samples and in a quantity proportional to the pellet volume. However, at this stage calcium dosage was more difficult to estimate accurately and both the purification efficiency and RNA recovery diminished by approx 20 to 30% (results not shown). In samples very rich in polymeric sugar, calcium treatment during the cell debris pelletting step appeared to be the most efficient in eliminating sugar. Moreover, calcium treatment seemed to be less efficient as fruits ripened, and determining the optimal dosage became more troublesome (results not shown). Volume 31, 2005 12-mm cross-diameter apple fruitlet Cortex Av. SD 122 ±13 1.9 ±0.16 1.9 ±0.17 100 Quantity (µg/g fw) A260/A230 A260/A280 Volume (µL) Seed Av. AZ SD 148 ±15 2 ±0.14 2.3 ±0.18 100 Av. Harvest time apple fruit Peduncle SD Av. SD Cortex Av. SD Skin Av. SD Harvest time peach fruit Seed Av. Mesocarp SD Av. SD Epicarp Av. SD 106 ±11 153 ±18 62 ±16 73 ±6 164 ±14 81 ±14 78 ±9 2.2 ±0.2 2.2 ±0.19 1.9 ±0.11 1.9 ±0.16 2 ±0.13 2 ±0.2 1.8 ±0.16 2 ±0.17 2 ±0.18 1.8 ±0.14 1.8 ±0.12 1.9 ±0.14 1.9 ±0.16 1.8 ±0.15 100 100 100 100 100 100 100 The quantity indicates the amount of RNA in microgram extracted from a gram of sample fresh weight. A260/A230 and A260/A280 are the purity indexes for sugars and proteins and the volume is the amount of water in microliters used to hydrate RNA pellet. Av and SD indicate, respectively, the average and standard deviation of at least five experiments. RNA Extraction From Plant Tissues MOLECULAR BIOTECHNOLOGY Table 2 Comparison Between Parameters in RNA Extracted From Different Samples 117 Table 3 Comparison Between the Amount of Calcium Solution Used for RNA Extraction for Different Samples 12-mm cross-diameter apple fruitlet 0.5 M calcium solution (µL) Harvest time apple fruit Harvest time peach fruit Cortex Seeds AZ Peduncle Apple root Apple leaf Cortex Skin Seeds Mesocarp Epicarp 50 20 120 140 35 40 100 120 20 20 50 117 Volume 31, 2005 The values indicate the volume of 0.5 M calcium stock solution in microliters added to extraction buffer per gram of sample to achieve high quality RNA as reported in Table 2. 118 Dal Cin et al. Fig. 2. Expression level of ubiquitin verified by semiquantitative RT-PCR (A) and Northern blotting (B) of RNA from peduncle extracted with ethylene glycol monobutyl ether (BE), calcium hydroxide (Ca(OH)2), or calcium chloride (CaCl2). Expression analysis with semiquantitative reverse transcriptase (RT)-PCR and Northern blotting indicated that when using the same quantity of RNA extracted with calcium hydroxide or calcium chloride or with BE there were very similar results in terms of RT efficiency and amplification (Fig. 2A) and in RNA migration and probe annealing (Fig. 2B). 4. Discussion We have described how a simple cation like Ca2+ may be used to improve RNA extraction. Its action is clearly visible by looking at the gel-like pellet at the bottom of the sample after cell debris precipitation. From our results it seems that it is the calcium itself and not its counter-ion that causes the precipitation of contaminating polymeric sugars, because calcium hydroxide and calcium chloride gave similar results in terms of RNA quality and yield. It is likely that calcium binds to carboxylic acid groups of demethylesterified chains of pectin, causing their aggregation and making it easier to precipitate them as a Ca2+–pectate gel. However, we have found that allowing calcium to act for several minutes can cause a decrease in RNA quality, probably as a result of degradation by nucleases, which require divalent cations as cofactors. Furthermore, the use of excess calcium brought about a decrease in RNA recovery, possibly due to aggregation and precipitation of nucleic acids (16). The best results MOLECULAR BIOTECHNOLOGY were seen for green tissues or early ripe fruit, probably because as fruits ripen there is a decrease in the size of polymeric pectins (17), making them more difficult to precipitate. Our results indicate that calcium can enhance RNA purity from different fruits and tissues but with a varying efficiency. A calcium precipitation step could be added to standard RNA extraction protocols where differential precipitations are performed, such as the hot-borate method (18 ). Acknowledgments We thank Dr. David A. Brummell of the Crop and Food Research, Fitzhebert Science Centre, New Zealand, for critical reading of the manuscript and for comments. References 1. Ainsworth, C. (1994) Isolation of RNA from floral tissue of Rumex acetosa (Sorrel). Plant Mol. Biol. Rep. 12,1 98–203. 2. Lopez-Gomez, R. and Gomez-Lim, M. A. (1992) A method for extracting intact RNA from fruits rich in polysaccharides using ripe mango mesocarp. Hort. Sci. 27, 440–442. 3. Mitra, D. and Kootstra, A. (1993) Isolation of RNA from apple skin. Plant Mol. Biol. Rep. 11, 326–332. 4. Wan, C. Y. and Witkins, T. A. (1994) A modified hot borate method significantly enhances the yield of high-quality RNA from cotton (Gossypium hirsutum L.). Anal. Biochem. 223, 7–12. 5. Su, X. and Gibor, A. 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(1996) Isolation of RNA from plant tissue. In: A Laboratory Guide to RNA: Isolation, Analysis, and Synthesis (Krieg, P. A., ed.). Wiley-Liss, New York, pp. 21–41. 13. Chang, S., Puryear, J., and Caurney, J. (1993) A simple and efficient method for isolating RNA from pine trees. Plant Mol. Biol. Rep. 11, 113–116. MOLECULAR BIOTECHNOLOGY 119 14. Ruperti, B., Bonghi, C., Rasori, A., Ramina, A., and Tonutti, P. (2001) Characterization and expression of two members of the peach 1-aminocyclopropane-1carboxylate oxidase gene family. Physiol. Plant 111, 336–344. 15. Quaggiotti, S., Ruperti, B., Pizzeghello, D., Francioso, O., Tugnoli, V., and Nardi, S. (2004) Effect of low molecular size humic substances on nitrate uptake and expression of genes involved in nitrate transport in maize (Zea mays L.). J. Exp. Bot. 55, 803–813. 16. Jackson, A. O. and Larkins, B. A. (1976) Influence of ionic strength, pH, and chelation of divalent metals on isolation of polyribosomes from tobacco leaves. Plant Physiol. 57, 5–10. 17. Brummell, D. A., Dal Cin V., Crisosto, C. H., and Labavitch, J. M. (2004) Cell wall metabolism during maturation, ripening and senescence of peach fruit. J. Exp. Bot. 55, 2029–2039. 18. Moser, C., Gatto, P., Moser, M., Pindo, M., and Velasco, R. (2004) Isolation of functional RNA from small amounts of different grape and apple tissues. Mol. Biotech. 26, 95–100. Volume 31, 2005