RNA isolation from siliques, dry seeds, and other tissues of Arabidopsis thaliana
2004; Future Science Ltd; Volume: 37; Issue: 4 Linguagem: Inglês
10.2144/04374bm03
ISSN1940-9818
AutoresYuji Suzuki, Tetsu Kawazu, Hiroyuki Koyama,
Tópico(s)Chromosomal and Genetic Variations
ResumoBioTechniquesVol. 37, No. 4 BenchmarksOpen AccessRNA isolation from siliques, dry seeds, and other tissues of Arabidopsis thalianaYuji Suzuki, Tetsu Kawazu & Hiroyuki KoyamaYuji SuzukiOji Paper Co. Ltd., Kameyama, MieGifu University, Gifu, Japan, Tetsu KawazuOji Paper Co. Ltd., Kameyama, Mie & Hiroyuki Koyama*Address correspondence to: Hiroyuki Koyama, Laboratory of Plant Cell Technology, Faculty of Agriculture, Gifu University, Gifu 501-1193, Japan. e-mail: E-mail Address: koyama@cc.gifu-u.ac.jpGifu University, Gifu, JapanPublished Online:6 Jun 2018https://doi.org/10.2144/04374BM03AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinkedInRedditEmail Arabidopsis thaliana is widely used in the research of plant molecular biology, but RNA isolation from certain tissues is difficult. For example, siliques and dry seeds require complex and time-consuming methods due to the high contents of polysaccharides and secondary metabolites (1–3). One way to treat such difficult tissues is to use a high-salt extraction buffer, an example being LiCl, which was used in previous studies (1,2). Here we report that the use of a high-sodium extraction buffer is more effective in RNA isolation from these tissues. Our method consists of a two-step extraction with organic solvents, isopropanol precipitation, and LiCl precipitation, if necessary, after sample extraction and is simple enough to be routinely applied to other tissues.Various tissues of soil-cultured or hydroponically cultured A. thaliana [eco-type Wassilewskija (Ws)] plants were used as samples. The extraction buffer contained NaCl and trisodium citrate at high concentrations, this combination being originally used for the isopropanol precipitation of RNA to reduce polysaccha-ride contamination (4). Sample homogenate was extracted with chloroform-isoamylalcohol prior to phenolchloroform-isoamylalcohol extraction to prevent contamination by polyphenolics (3,5). The phenol mixture was based on the acid guanidium-phenol-chloroform (AGPC) method (6) and was used to remove protein and DNA from the RNA fraction. LiCl precipitation can also be conducted if polysaccharide contamination occurs (for example, when heavily stressed samples are used).An aliquot of total RNA was diluted with a Tris-EDTA buffer [1 mM Tris-HCl (pH 8.0), 0.1 mM EDTA (pH 8.0)], and its RNA concentration was measured by its absorbance at 260 nm (A260). The yields of total RNA from siliques and dry seeds were 286 ± 20 and 1104 ± 75 µg/g tissue, respectively. These values were within or above the range of RNA yield in the previous reports (2,3). The yields in other tissues such as flower buds, leaves, roots, and stems were 1159 ± 135, 550 ± 108, 513 ± 91, and 392 ± 42 µg/g tissue, respectively. The ratios of A260/A280 and A260/A230 of these samples were 2.13–2.17 and 2.39–2.58, respectively, indicating no severe contamination by protein or polysaccharides. DNA contents of the RNA fractions with or without DN-ase I (TAKARA BIO, Ohtsu, Shiga, Japan) treatment were measured with the Fluorescent DNA Quantitation kit (Bio-Rad Laboratories, Hercules, CA, USA). The amounts of contaminating DNA in all the RNA fractions prepared using this method were similar to those measured in samples prepared with the AGPC method from shoots and roots of Arabidopsis. The results of the electropherogram of the RNA showed clear peaks of ribosomal RNAs (rRNAs) (Figure 1).Figure 1. Electropherogram of the total RNA isolated from various tissues of Arabidopsis thaliana.An aliquot of total RNA was analyzed with Model 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA). Panels (A) dry seeds, (B) siliques, (C) flower buds, (D) leaves, (E) roots, and (F) stems.An aliquot (1 µg) of the DNase I-treated total RNA was reverse-transcribed with the SuperScript™ First Strand Synthesis System (Invitrogen, Carlsbad, CA, USA) for reverse transcription PCR (RT-PCR). DNA fragment of polyubiquitin, whose transcript level is low (UBQ4; GenBank® accession no. NM_122069) (7), was then amplified with TaKaRa Taq™ (TAKARA BIO). The primer pair used was 5′-CCTAGATCGCTCTTCACATCTC-3′ and 5′-GCGGAATGTTTTAACATGCACT-3′. The PCR conditions were 26 cycles with 30 s at 94°C, 30 s at 54°C, and 90 s at 72°C. A DNA fragment at a predicted molecular weight (about 1.4 kb) was amplified (Figure 2). These results indicate that the total RNA fractions remain intact and can be applied to enzymatic reactions.RNA Isolation ProtocolReagentsExtraction buffer: 100 mM Tris-HCl (pH 9.5), 10 mM EDTA (pH 8.0), 2% (w/v) lithium dodecyl sulfate, 0.6 M NaCl, and 0.4 M trisodium citrate. Before use, add 2-mercaptoethanol to obtain a final concentration of 5% (v/v).Phenol mixture: water-saturated phenol containing 35% (w/v) of guanidium thiocyanate and 1/10 vol of 2 M sodium acetate (pH 4.0). Store at 4°C.ProcedureGrind the tissue to a fine powder in liquid nitrogen using a mortar and pestle. A small amount of acid-washed quartz sand should be added when dry seeds are used as samples.Transfer the sample powder to a tube containing a 20 vol (dry seeds) or 5–10 vol (other tissues) of the extraction buffer and mix immediately. Using a 50-mL tube is recommended because of the facility for the mixing even if the sample volume was small (about 50 mg of dry seeds, for example). A chemical hood can be used to avoid the strong odor caused by 2-mercaptoethanol. Transfer the homogenate to 1.5- or 2.0-mL tubes. Centrifuge at ≥12,000× g for 5 min at room temperature. Transfer the supernatant to 1.5- or 2.0-mL tubes.Add 1 vol of chloroform:isoamylalcohol (CIA; 24:1). Mix by inversion by hand 15 times. Centrifuge at ≥12,000× g for 5–10 min at 4°C. Transfer the upper phase to 1.5- or 2.0-mL tubes. Contamination by the middle phase was allowed to some extent but not recommended.Add 1 vol of the phenol mixture. Mix and incubate for 3 min at room temperature. Add CIA at a 1/2 vol of the phenol mixture. Shake vigorously for 15 s by hand. Centrifuge at ≥12,000× g for 5 min at 4°C. Transfer the upper phase to 1.5-mL tubes.Add 0.6 vol of isopropanol. Mix and incubate for 10 min at room temperature. Centrifuge at ≥12,000× g for 15 min at 4°C.Wash the pellet with 75% (v/v) ethanol. Air-dry briefly. Dissolve in a small amount of RNase-free water. Store at −80°C.Optional: when dry seeds are used as samples, add 1/3 vol of 8 M LiCl. Mix and incubate for ≥2 h at −80°C. Melt and centrifuge at ≥12,000× g for 30 min at 4°C.Dissolve the pellet in a small amount of RNase-free water. Add 1/10 vol of 3 M sodium acetate (pH 5.2) and 1 vol of isopropanol. Incubate for 10 min at room temperature. Centrifuge at ≥12,000× g for 15 min at 4°C.Wash the pellet with 75% (v/v) ethanol. Air-dry briefly. Dissolve in a small amount of RNase-free water. Store at −80°C.Figure 2. Reverse transcription PCR (RTPCR) amplification of mRNA of UBQ4 using the total RNA isolated from various tissues of Arabidopsis thaliana as a template.Lanes M, molecular weight marker; 1, dry seeds; 2, siliques; 3, flower buds; 4, leaves; 5, roots; 6, stems.This method drastically reduces the number of steps of sample treatment and the time required (at least less than a half) in comparison with the previous methods (1–3). It also facilitates RNA isolation from other plant tissues, such as cultured cells of rice and carrots, which contain a large amount of polysaccharides.AcknowledgmentsThis study was supported by a grant from the New Energy and Industrial Technology Development Organization (NEDO).Competing Interests StatementThe authors declare no conflicts of interest.References1. Carpenter, C.D. and A.E. Simon. 1998. Preparation of RNA, p. 85–89. In J. Martinez-Zapater and J. Salinas (Eds.), Methods in Molecular Biology, vol. 82, Arabidopsis Protocols. Humana Press, Totowa.Crossref, Google Scholar2. Vicient, C.M. and M. Delseny. 1999. Isolation of total RNA from Arabidopsis thaliana seeds. Anal. Biochem. 268:412–413.Crossref, Medline, CAS, Google Scholar3. Ruuska, S.A. and J.B. Ohlrogge. 2001. Protocol for small-scale RNA isolation and transcriptional profiling of developing Arabidopsis seeds. BioTechniques 31:752–758.Link, CAS, Google Scholar4. Chomczynski, P. and K. Mackey. 1995. Modification of the TRI reagent procedure for isolation of RNA from polysaccharide- and proteoglycan-rich sources. BioTechniques 19:942–945.Medline, CAS, Google Scholar5. Schultz, D.S., R. Craig, D.L. Cox-Foster, R. Mumma, and J.I. Medford. 1994. RNA isolation from recalcitrant plant tissues. Plant Mol. Biol. Rep. 12:310–316.Crossref, CAS, Google Scholar6. Chomczynski, P. and N. Sacchi. 1987. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162:156–159.Crossref, Medline, CAS, Google Scholar7. Sun, C.W. and J. Callis. 1997. Independent modulation of Arabidopsis thaliana polyubiquitin mRNAs in different organs and in response to environmental changes. 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