Functional RNA From Grape and AppleRESEARCH Tissues
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Isolation of Functional RNA From Small Amounts of Different Grape and Apple Tissues Claudio Moser,* Pamela Gatto, Mirko Moser, Massimo Pindo, and Riccardo Velasco Abstract An efficient, simple, and small-scale procedure for isolating functional ribonucleic acid (RNA) was successfully applied to many different tissues of grape and apple. These woody plants are rich in polyphenolic compounds and polysaccharides that could impair the RNA extraction. The method chosen is based on the use of hot borate buffer at alkaline pH supplemented with several adjuvants and followed by selective precipitations. Starting with only 0.4 g of fresh tissue and working with small tubes (2 mL), we were able to obtain good yields of high-quality RNA suitable for further applications. The procedure can be proposed for many applications, and it is particularly highly recommended when isolating RNA from a large number of samples. Index Entries: Apple; grape; hot borate; RNA contamination; RNA extraction.
1. Introduction Isolation of high-quality ribonucleic acid (RNA) from woody plants is often a difficult task. The generally high content of secondary metabolites and polysaccharides in their tissues hinders the purification process, interferes with absorbancebased quantification, and can inhibit downstream enzymatic manipulations (1). The high number of secondary metabolites and polysaccharides and their relative abundance, depending on species and tissues, are the reasons underlying the development of different protocols for each specific RNA extraction (2–7). In this regard, important crops like grape (Vitis vinifera L.) and apple (Malus spp.) are not an exception: many procedures for RNA extraction have been published either for grape (8–11) or for apple (12–14). However, these methods, are not completely satisfying because they sometimes yield low-quality RNA, and they require ultracentrifugation steps or the use of phenol (8–14). A method based on the use of hot borate and proteinase K was devel-
oped to isolate total RNA from bean seeds (15) and then modified by addition of a few adjuvants to isolate total RNA from cotton leaves and other plant species (16). Another method based on the use of mixed detergents and high ionic strength has been described to isolate total RNA from conifer species (17). Both these procedures do not involve the use of phenol and ultracentrifugation. Here we report the successful application of the hot borate method to the isolation of high-quality total RNA from many different tissues of grape and apple. This method is particularly recommended with a limited amount of starting material.
2. Materials and Methods 2.1. Plant Materials Vitis vinifera L. “Pinot noir” and the Malus sieboldii (Regel) Rehd. tissues were collected from plants in the field collections of the Istituto Agrario di San Michele all’Adige, Trento, Italy, and immediately frozen in liquid nitrogen. Samples were stored at –80°C until extraction.
*Author to whom all correspondence and reprint requests should be addressed: Dr. Claudio Moser, Istituto Agrario di S. Michele all’Adige, I38010, S. Michele a/Adige, Trento-Italy. Fax: +390461650956. E-mail:
[email protected] Molecular Biotechnology 2004 Humana Press Inc. All rights of any nature whatsoever reserved. 1073–6085/2004/26:2/95–99/$25.00
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2.2. Reagents 1. Extraction buffer (XT buffer): 0.2M sodium borate decahydrate, 0.03M ethylenediaminetetraacetic acid (EDTA) pH 8, 1% (w/v) sodium dodecyl sulfate (SDS), 1% (w/v) deoxycholate sodium salt, 2% (v/v) β-mercaptoethanol, 0.5% (w/v) spermidine, 1% (w/v) Igepal (Sigma), 2% (w/v) PVP-40 (mol wt 40,000; see Note 1). To dissolve, add components to prewarmed (50°C) RNase-free H2O and adjust the pH to pH 9.0 with NaOH. 2. Proteinase K at 20 mg/mL. 3. 0.01M Tris-HCl, pH 7.5. 4. 2M KCl. 5. 2M potassium acetate, pH 5.5. 6. 2M and 8M ice-cold LiCl. 7. 80% (v/v) ethanol. 8. Isopropanol.
2.3. RNA Extraction Protocol We experienced that using disposable 2-mL Eppendorf tubes instead of larger tubes throughout the procedure improved reproducibility and RNA yield. 1. Grind the frozen tissue to a fine powder with a prechilled mortar and pestle, adding liquid nitrogen to tissue as needed. 2. Using a prechilled spatula, put 0.4 g of the powdered tissue into a 2-mL Eppendorf tube, and immediately add 1.4 mL of prewarmed (80°C) XT buffer. Mix thoroughly and keep it for 5 min at 80°C. 3. Add 1 mg of proteinase K to each sample, and incubate 1 h at 42°C with gentle shaking. 4. Add 2M KCl to a final concentration of 160 mM. Mix and keep the tube on ice for 45 min (see Note 2). 5. Centrifuge at 15,000g for 15 min at 8°C to remove debris. 6. Transfer the supernatant to a new 2-mL Eppendorf tube, and add 1/3 vol. of 8M LiCl (to a final concentration of 2M) and β-mercaptoethanol to a final concentration of 1% (v/ v). Incubate on ice at 4°C overnight. 7. Collect precipitated RNA by centrifugation at 15,000g for 25 min at 8°C. Discard supernatant. 8. Wash the pellet with 1 mL of cold LiCl 2M, and then centrifuge at 15,000g for 15 min at 8°C. Discard supernatant.
9. Repeat step 8 in Subheading 2.3. if the supernatant is still pigmented. 10. Suspend the pellet with 600 µL of 10 mM TrisHCl pH 7.5, mixing thoroughly at room temperature. 11. Add 1/10 vol of 2M potassium acetate, pH 5.5; mix and incubate 10 min on ice (see Note 3). 12. Centrifuge at 15,000g for 15 min at 8°C to remove insoluble material. Transfer the RNA containing supernatant into a new tube. 13. Precipitate the RNA by adding 0.9 vol of cold isopropanol. Incubate 1 h at –20°C and then centrifuge at 15,000g for 25 min at 8°C. Discard the supernatant. 14. Wash the pellet in 1 mL cold 80% ethanol, and collect the RNA by centrifugation at 15,000g for 10 min at 8°C. 15. Aspirate ethanol and dry the pellet. Dissolve it in 50–100 µL RNase-free ddH2O.
2.4. Estimation of RNA Purity Quality and quantity of the RNA samples were evaluated by electrophoresis in TBE-agarose gels stained with ethidium bromide and by measurements of the absorbance spectrum between 210 and 310 nm.
2.5. RT–PCR The grape RNA samples were treated with DNase I (RNase-free, Promega) to eliminate eventual genomic DNA contamination. After enzyme removal, 1–5 µg of RNA samples were amplified by a two-step reverse transcription polymerase chain reaction (RT–PCR) method using the Superscript II RNase H-RT enzyme (Invitrogen). First-strand cDNA synthesis was performed in a 20-µL reaction volume at 42°C for 50 min, priming the template with 0.5 µg of oligo (dT)12–18 0.5 mM deoxynucleo–tide-triphosphate (dNTP) mix, 10 mM dithio–threitol (DTT), 1X first-strand buffer (25 mM Tris-HCl pH 8.3, 37.5 mM KCl, 1.5 mM MgCl2), 1 µL (40 U) of RNaseOUT ribonuclease inhibitor (Invitrogen), and 200 U of Superscript II RT. After heat inactivation of the RT enzyme (15 min at 70°C), 5 µL of the first-strand complementary DNA (cDNA) reaction were used to amplify specific internal regions of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and of the mono-oxygenase
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Fig. 1. Gel electrophoresis of total RNA samples. Visualization of total RNA isolated from different tissues run on 1% agarose gel stained with ethidium bromide. Grape: root (lane 1), berry (lane 2), leaf (lane 3), bud (lane 4), flower (lane 5); apple: leaf (lane 6), phloematic tissue (lane 7). Lane M1: RNA ladder (Genenco), lane M2: MassRuler DNA ladder low range (Fermentas).
cDNAs with the grape and the apple samples, respectively. In addition to the cDNA template, the PCR reaction (50 µL total vol) contained 0.2 mM dNTP mix, 1.5 mM MgCl 2, 0.2 µM of each primer, and 2 U of Taq DNA polymerase supplied with its own buffer. The PCR mixture was initially denatured at 94°C for 2 min, and then subjected to 38 cycles at the following conditions: 94°C for 45 s, 55°C for 45 s, 72°C for 60 s, with a final extension at 72°C for 7 min. PCR products (20% of total reaction) were analyzed on 1.2% TBE-agarose gel stained with ethidium bromide.
3. Notes 1. Add β-mercaptoethanol, spermidine, Igepal, and PVP-40 immediately before use. 2. A precipitation of proteins and other material is sometimes observed following KCl addition. 3. This step is highly recommended to improve RNA quality when starting from grape berries.
4. Results and Discussion Isolation of total RNA from grape and apple tissues by extraction with phenol and high-molarity guanidinium salts (18) or TRIzol (Invitrogen) gives unsatisfactory results probably because of the high levels of phenolic compounds and polysaccharides (10, personal experience and communication of Matteo Komjanc). A method
based on hot borate, proteinase K, and a few adjuvants is highly recommended to isolate high-quality RNA from recalcitrant plant tissues containing copious levels of secondary metabolites (1). This protocol uses an extraction buffer including a high concentration of borate, which makes hydrogenbonds with the polyphenolics compounds and contains proteinase K to inactivate RNases. Introducing few important changes, we successfully applied this method to isolate high-quality RNA from five grape and two apple tissues. Electrophoretic analysis in agarose gel revealed indeed a good integrity of the samples and almost no genomic DNA contamination (Fig. 1). Quality and yield were also judged by spectrophotometric measurements and are summarized in Table 1. The A260/A280 and A260/A230 ratios ranged for almost all the samples between 1.8 and 2.4, indicating no major contamination from proteins, polysaccharides or polyphenols. The apple samples showed a slightly lower value for the A260/A280 ratio. Although not needed for an efficient reverse transcription, an additional ethanol precipitation of the RNAs could improve the absorbance ratio (data not shown). Yields are quite different among tissues. In grape, yields are the highest in leaves (ca. 0.8 mg/gfw), intermediate in buds and flowers (ca. 0.4 mg/gfw), and lowest in roots and berries (0.160 mg/gfw and 0.035 mg/gfw,
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Moser et al. Table 1 Spectrophotometric Measurement of Isolated RNAs to Evaluate Purity (A260/230 and A260/280) and Yield (µg/gfw) Species
Tissue
A260/A230
A260/A280
Yield (µg/gfw)
Root Berry Leaf Bud Flower
1.89 (0.06) 1.80 (0.01) 2.41 (0.06) 2.31 (0.00) 2.22 (0.05)
1.88 (0.01) 1.83 (0.03) 1.97 (0.14) 2.00 (0.01) 1.99 (0.02)
163 (188.3)* 28 (1.7) 818 (170.4) 488 (200.6) 389 (110.9)
Leaf Phloem
2.67 (0.04) 2.45 (0.26)
1.74 (0.03) 1.75 (0.09)
540 (238.1) 1088 (554.4)
Vitis
Malus
Note: Results are the mean of three samples (standard deviation). *The high standard deviation is probably due to the heterogenity of the sample: young and older root.
Fig. 2. Gel electrophoresis of RT–PCR products. RT–PCR products obtained using gene-specific primers run on 1.2% agarose gel stained with ethidium bromide. GAPDH amplification: flower (lane 1), leaf (lane 2), berry (lane 3), root (lane 4), bud (lane 5). Mono-oxygenase amplification: leaf (lane 6), phloem (lane 7). M: MassRuler DNA ladder low range, (Fermentas). Negative controls (no RT-enzyme added) did not show any amplification product (data not shown).
respectively). In apple, yields of extraction from phloematic tissues (ca. 1 mg/gfw) are approximately twice the yields from leaves (ca. 0.54 mg/ gfw). These differences are consistent with the structural and metabolic properties of the distinct tissues. The amount of total RNA per gram of fresh weight obtained with the described procedure is comparable to or better than the amounts reported in the literature (8,10). Suitability of the RNA for downstream applications was demonstrated by reverse transcription and PCR amplification by the use of gene-specific primers (Fig.
2). Further validation of the RNA quality comes from the construction of cDNA libraries starting from this material. Changes to the original method (1), although apparently very small, enabled, in our hands, major improvements in reproducibility. They involved the use of 2-mL disposable tubes instead of larger tubes, the addition of β-mercaptoethanol during the lithium chloride overnight precipitation, and the omission of the last precipitation step. We noticed that working with small tubes allowed us to centrifuge at higher speed for a few
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Functional RNA From Grape and Apple Tissues minutes, giving more reproducible results in a shorter time; β-mercaptoethanol was added to reduce degradation of the RNA by any RNases. In conclusion, the procedure described yields good quantities of high-quality RNA when used on grape and apple tissues, even if working with a small amount of material. For this reason and for its simplicity (no special equipment or kits needed), this method appears particularly suitable to process a large number of samples at low cost.
Acknowledgments We wish to thank Matteo Komjanc for supplying the mono-oxygenase specific primers, Jose Vouillamoz for critical reading of the manuscript, and Maria Piques for her kind help. This work was supported by the project Advanced Biology funded by the Fondazione delle Casse di Risparmio di Trento e Rovereto (Trento-Italy). P.G., M.M. and M.P. are recipients of a fellowship granted by Fondo Unico of the Provincia di Trento, Resveratrol, SMAP, and BAC-co, respectively.
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