BENCHMARKS RNA isolation from siliques, dry seeds, and other tissues of Arabidopsis thaliana

Yuji Suzuki1,2, Tetsu Kawazu1, and Hiroyuki Koyama2

1Oji

Paper Co. Ltd., Kameyama, Mie and 2Gifu University, Gifu, Japan

BioTechniques 37:542-544 (October 2004)

Arabidopsis thaliana is widely used method (6) and was used to remove in the research of plant molecular biolprotein and DNA from the RNA fracogy, but RNA isolation from certain tistion. LiCl precipitation can also be consues is difficult. For example, siliques ducted if polysaccharide contaminaand dry seeds require complex and tion occurs (for example, when heavily time-consuming methods due to the stressed samples are used). high contents of polysaccharides and An aliquot of total RNA was disecondary metabolites (13). One way luted with a Tris-EDTA buffer [1 mM to treat such difficult tissues is to use a Tris-HCl (pH 8.0), 0.1 mM EDTA (pH high-salt extraction buffer, an example 8.0)], and its RNA concentration was being LiCl, which was used in previous measured by its absorbance at 260 nm studies (1,2). Here we report that the (A260). The yields of total RNA from use of a high-sodium extraction buffer siliques and dry seeds were 286 20 is more effective in RNA isolation from and 1104 75 g/g tissue, respectively. these tissues. Our method consists of a These values were within or above the two-step extraction with organic solrange of RNA yield in the previous revents, isopropanol precipitation, and ports (2,3). The yields in other tissues LiCl precipitation, if necessary, after such as flower buds, leaves, roots, and sample extraction and is simple enough stems were 1159 135, 550 108, 513 to be routinely applied to other tissues. 91, and 392 42 g/g tissue, respecVarious tissues of soil-cultured or tively. The ratios of A260/A280 and A260/ hydroponically cultured A. thaliana [ecotype 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 polysaccharide 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 Figure 1. Electropherogram of the total RNA isolated from various tissues of Arabidopsis thaliana. An aliquot of total g u a n i d i u m - p h e n o l - RNA was analyzed with Model 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA). Panels (A) dry seeds, (B) chloroform (AGPC) siliques, (C) flower buds, (D) leaves, (E) roots, and (F) stems.
542 BioTechniques Vol. 37, No. 4 (2004)

A230 of these samples were 2.132.17 and 2.392.58, respectively, indicating no severe contamination by protein or polysaccharides. DNA contents of the RNA fractions with or without DNase 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). An aliquot (1 g) of the DNase Itreated 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 5CCTAGATCGCTCTTCACATCTC-3 and 5-GCGGAATGTTTTAACATGCACT-3. The PCR conditions were 26 cycles with 30 s at 94C, 30 s at 54C,

BENCHMARKS
RNA Isolation Protocol Reagents Extraction 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 4C. Procedure 1. Grind 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. 2. Transfer the sample powder to a tube containing a 20 vol (dry seeds) or 510 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. 3. Add 1 vol of chloroform:isoamylalcohol (CIA; 24:1). Mix by inversion by hand 15 times. Centrifuge at 12,000 g for 510 min at 4C. 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. 4. 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 4C. Transfer the upper phase to 1.5-mL tubes. 5. Add 0.6 vol of isopropanol. Mix and incubate for 10 min at room temperature. Centrifuge at 12,000 g for 15 min at 4C. 6. Wash the pellet with 75% (v/v) ethanol. Air-dry briefly. Dissolve in a small amount of RNase-free water. Store at -80C. 7. Optional: when dry seeds are used as samples, add 1/3 vol of 8 M LiCl. Mix and incubate for 2 h at -80C. Melt and centrifuge at 12,000 g for 30 min at 4C. 8. 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 4C. 9. Wash the pellet with 75% (v/v) ethanol. Air-dry briefly. Dissolve in a small amount of RNase-free water. Store at -80C.

and 90 s at 72C. 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. 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 (13). It also facilitates RNA isolation from other plant tissues, such as cultured cells of rice and carrots, which contain a large amount of polysaccharides.

ACKNOWLEDGMENTS

This study was supported by a grant from the New Energy and Industrial Technology Development Organization (NEDO).
COMPETING INTERESTS STATEMENT

R. Mumma, and J.I. Medford. 1994. RNA isolation from recalcitrant plant tissues. Plant Mol. Biol. Rep. 12:310-316. 6.Chomczynski, P. and N. Sacchi. 1987. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162:156-159. 7.Sun, C.W. and J. Callis. 1997. Independent modulation of Arabidopsis thaliana polyubiquitin mRNAs in different organs and in response to environmental changes. Plant J. 11:1017-1027.

The authors declare no conflicts of interest.

REFERENCES
1.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. 2.Vicient, C.M. and M. Delseny. 1999. Isolation of total RNA from Arabidopsis thaliana seeds. Anal. Biochem. 268:412-413. 3.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. 4.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. 5.Schultz, D.S., R. Craig, D.L. Cox-Foster,

Received 24 May 2004; accepted 29 June 2004. Address correspondence to Hiroyuki Koyama, Laboratory of Plant Cell Technology, Faculty of Agriculture, Gifu University, Gifu 501-1193, Japan. e-mail: koyama@cc. gifu-u.ac.jp

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. 544 BioTechniques

Vol. 37, No. 4 (2004)