J Appl Phycol (2009) 21:233238 DOI 10.

1007/s10811-008-9354-0

Improved RNA isolation from Laminaria japonica Aresch (Laminariaceae, Phaeophyta)

Jianting Yao & Wandong Fu & Xiuliang Wang & Delin Duan

Received: 18 March 2008 / Revised and accepted: 20 May 2008 / Published online: 1 August 2008 # Springer Science + Business Media B.V. 2008

Abstract RNA isolation is difficult in some plants and algae because phenolics, polysaccharides, or other compounds can bind or co-precipitate with RNA, and because the success of RNA isolation can be strain-specific and species-specific. To create an improved RNA isolation protocol for Laminaria japonica Aresch (Laminariaceae, Phaeophyta), four methods for extracting RNA were tested. A cetyltrimethylammonium bromide (CTAB)-based RNA extraction protocol was developed that clearly showed 28S and 18S ribosomal RNA bands and produced RNA with high yield (68 g g1 fresh weight) and high quality (A260/280 ratio 1.960.05). The isolated RNA was intact, and RT-PCR analysis confirmed that further molecular application is feasible. Keywords Phaeophyta . Phenolics . CTAB . Polysaccharides . RNA extraction

Introduction Laminaria japonica Aresch (Laminariales, Phaeophyta) grows in temperate cold-water zones and is native to the northwest coasts of the Pacific Ocean. The species was introduced to China in 1927 from Hokkaido, Japan. Tseng et al. (1955) devised a method based on collecting zoospores in early summer that led L. japonica to become a large-scale cultivation industry in China. Today, L. japonica is used in China mainly as a foodstuff and raw
J. Yao : W. Fu : X. Wang : D. Duan (*) Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, China e-mail: dlduan@ms.qdio.ac.cn J. Yao : W. Fu Graduate School of the Chinese Academy of Sciences, Beijing 100059, China

material for iodine, mannitol and alginate production (Tseng and Qin 1999). Annual production in China is about 900,000 t dry Laminaria and 13,000 t algin (Tseng 2001). Improved cultivation and use of L. japonica requires genetic studies based on isolating high-quality RNA for applications such as northern blotting, macroarray hybridization, reverse transcription-polymerase chain reaction (RT-PCR) analysis, and cDNA library construction (Baker et al. 1990; Graham 1993; Lewinsohn et al. 1994; Portillo et al. 2006; Wang et al. 2008). Various factors inhibit isolation: RNA molecules are subject to enzymatic degradation by RNase, and seaweed tissues have high levels of phenolic compounds, polysaccharides and secondary metabolites after cells embedded in viscous polysaccharides are disrupted (Ho et al. 1996; Li et al. 2006; Vanessa et al. 2008); phenolic compounds oxidize readily, link covalently with quinones and bind to nucleic acids (Loomis 1974); and polysaccharides co-precipitate with RNA in low ionic strength buffers (Noonberg et al. 1995; Su and Gibor 1988; Birtic and Kranner 2006). Samples from different species, organs or parts at different times of the year have slightly different cell wall compositions (Dring 1982). There are many reports of successful RNA isolation for different species, and for the same species grown in different environments (Hong et al. 1997; Gehrig et al. 2000; Chan et al. 2004; Shi and Bressan 2006). Some RNA extraction protocols for plants rich in phenolics and polysaccharides use sodium dodecyl sulfate (SDS), soluble polyvinylpyrrolidone (PVP) and ethanol precipitation (Dong and Dunstan 1996). One protocol modifies an acid guanidinium thiocyanate-phenol-chloroform method (Chomczynski and Sacchi 1987), and another modifies the hot borate method (Wan and Wilkins 1994); one protocol uses acetone treatment of freeze-dried and powdered plant materials (Schneiderbauer et al. 1991) and another modifies the cetyltrimethylammonium bromide (CTAB) method (Apt

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et al. 1995; Gareth et al. 2006; Wang et al. 2008). Several commercial kits for RNA extraction are used for isolating high quality RNA from some higher plants. In an effort to determine the best possible application for L. japonica, four RNA extraction methods were compared. Based on testing of these methods, an improved RNA extraction protocol was formulated.

Materials and methods Fresh samples of Laminaria japonica were collected from Jiaozhou Bay in QingDao, China in January 2007. Plant material was young, healthy and had relatively few epiphytes. Samples were washed with filtered seawater and rinsed in deionized distilled water to eliminate epiphytes before immediate freezing in liquid nitrogen and storage at 80C. Total RNA extraction Nondisposable plastic material was treated with 0.1% DEPC-treated water and autoclaved. Glass material and the mortar and pestle were treated for 4 h at 200C. Frozen tissues were ground in liquid nitrogen and RNA was extracted by four methods. Method 1: modified from Chomczynski and Sacchi 1987 Ground frozen tissue (approximately 0.5 g weight per sample) was added to 2 mL extraction buffer [4 M guanidine thiocyanate (Sangon, Shanghai, China), 25 mM sodium citrate pH 7.0, 0.5% (w/v) N-lauryl sarcosine (Sangon), 1% (v/v) mercaptoethanol (Sangon)]; 200 L 3 M sodium acetate (pH 4.8) was added to the mixture and mixed thoroughly, then 1 mL phenol/chloroform/isoamyl alcohol (25:24:1, v/v/v) was added and mixed thoroughly. The final suspension was vigorously agitated for 10 s and cooled on ice for 15 min, followed by centrifugation at 10,200 g for 20 min at 4C. To precipitate RNA, the aqueous phase was transferred to a fresh tube, mixed with 2 mL isopropanol and stored at 20C for 1 h. The tube was centrifuged at 10,200 g for 20 min at 4C. The RNA pellet was dissolved in 300 L extraction buffer and transferred to a 1.5 mL Eppendorf tube. The mixture was precipitated with 1 vol isopropanol at 20C for 1 h and centrifuged at 10,200 g for 10 min at 4C. The RNA pellet was washed with 75% (v/v) ethanol, dissolved in 40 L RNase-free water and stored at 80C. Method 2: modified from Dong and Dunstan 1996 Frozen tissue (approximately 1.0 g weight per sample) was ground in liquid nitrogen and transferred with a pre-cooled

spatula to a 50 mL tube containing 15 mL extraction buffer [50 mM Tris-HC1 (pH 8.0), 300 mM NaCl, 5 mM EDTA, 2% (w/v) SDS, 0.5% (w/v) soluble polyvinylpyrrolidone (Sangon)]. The mixture was vigorously mixed and incubated in a waterbath at 65C for 10 min with occasional agitation. The mixture was transferred to a fresh 50 mL tube and centrifuged for 15 min at 10,200 g at 4C. The supernatant was transferred to a fresh tube and 0.7 mL 3 M potassium acetate (pH 4.8) was added and thoroughly mixed. The tube was placed on ice for 30 min, followed by centrifugation at 10,200 g for 10 min at 4C. The supernatant was transferred to a fresh 50 mL tube, 5 mL 8 M lithium chloride was added and the mixture was left overnight at 4C. The tube was centrifuged at 10,200 g for 30 min at 4C. The supernatant was removed and the pellet was washed twice with 10 mL 3 M sodium acetate (pH 5.2) by resuspending the pellet and centrifuging at 10,000 g for 10 min at 4C. The final pellet was dissolved in 5 mL RNase-free glass distilled water. Crude total RNA solution was extracted using equal 5 mL phenol (saturated with TrisHCl, pH 8.0), 5 mL phenol/chloroform/isoamyl alcohol (25:24:1, v/v/v) and 5 mL chloroform/isoamyl alcohol (24:1, v/v). The mixture was centrifuged at 10,000 g for 10 min at 4C. The upper aqueous phase was transferred to a fresh tube and 0.5 ml 3 M sodium acetate (pH 5.2) and 13 mL absolute ethanol was added. The mixture was left at 80C for 2 h, centrifuged at 10,200 g for 20 min at 4C and again at 10,200 g for 10 min at 4C. The pellet was washed once with 75% (v/v) ethanol and air-dried for about 10 min. The RNA pellet was dissolved in 40 l RNase-free water and stored at 80C. Method 3: following the manual for the UNIQ-10 column Trizol total RNA extraction kit (Sangon) Ground frozen tissue (approximately 0.15 g weight per sample) was added to 0.5 mL Trizol solution, mixed thoroughly by pipetting and transferred to a 1.5 mL RNasefree Eppendorf tube. Chloroform/isoamyl alcohol (100 L; 24:1, v/v) was added and the suspension was vigorously agitated by shaking for 30 s, then centrifuged for 5 min at 12,000 g at room temperature. The supernatant was placed in a fresh tube and 0.15 mL absolute ethanol was added and mixed thoroughly. The mixture was transferred to the 2 mL collection tube supplied in the kit, incubated at room temperature for 2 min, centrifuged at room temperature for 1 min at 8,000 g and the flow-through was discarded. RPE solution (450 L) was added to the column, the tube was centrifuged for 30 s at 10,000 g at room temperature and the flow-through was discarded. The tube was centrifuged for 30 s at 10,000 g at room temperature. The tube was transfered to a fresh 1.5 mL RNase-free Eppendorf tube, 40 L RNase-free water was added and the tube was

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incubated in a waterbath at 55C for 2 min and centrifuged for 1 min at 8,000 g at room temperature. The solution was stored in an 1.5 mL Eppendorf tube at 80C. Method 4: modified from Apt et al. 1995 Ground frozen tissue (approximately 0.5 g weight per sample) was combined with 2 mL extraction buffer [100 mM Tris, 50 mM EDTA, pH 7.5, 2 M NaCl and 2% (w/v) CTAB (Sangon)]; 100 L 2 M DTT (TaKaRa, Kyoto, Japan) was added as antioxidant. The mixture was placed for 15 min at room temperature and then a one-quarter volume of absolute ethanol and one-ninth volume of 3 M potassium acetate (pH 4.8) was added slowly and mixed by gentle agitation. The mixture was extracted with 2 mL chloroform: isoamyl alcohol (24:1 v/v), vortexed vigorously, and centrifuged at 10,200 g for 25 min at 4C. The upper aqueous phase was transferred to a new tube. RNA was precipitated with one-tenth volume of 3 M sodium acetate (pH 5.0) and two volumes of pure ethanol in the presence of 4% (v/v) -mercaptoethanol antioxidant. Precipitation was completed in 30 min at 80C. RNA was collected by centrifugation at 14,000 g for 30 min at 4C. The pellet was washed with 75% ethanol and centrifuged at 14,000 g for 10 min at 4C. After drying (5 10 min at room temperature), the RNA was resuspended in 40 L RNase-free water and stored at 80C. Quantitative and qualitative analysis Quantitative analysis of RNA was by measuring optical density at 260 nm and 280 nm using a biophotometer (Eppendorf, Germany), with one unit of absorption at 260 nm representing 40 g mL1 RNA. Polysaccharide contamination was determined by maximum absorbance measurement at 230 nm. Ratio measurements at wavelengths 230, 260 and 280 indicated degree of RNA purity, and a pure RNA sample was indicated by a ratio of 1:2:1 at 230, 260 and 280 nm, respectively. The absorption ratio A260/230 indicated polysaccharide/polyphenolic contaminants and A260/280 indicated protein contaminates (Asif et al. 2000; Logemann et al. 1987; Manickavelu et al. 2007; Manning 1990). Total RNA solutions were loaded on a 1.5% agarose gel, electrophoresed to separate RNA, stained with ethidium bromide (EtBr), and visualized under UV light to assess the integrity of ribosomal bands. RT-PCR analysis To test RNA quality, 15 g total RNA was treated with DNase I, RNase-free (Fermentas, Hanover, MD) in a 30 L reaction containing 50 mM MgCl2 and 10 L template RNA, and the mixture was incubated for 30 min at 37C. Effective DNase I inactivation was achieved by adding 3 L 25 mM

EDTA to samples and incubating the reaction mix at 37C for 1 min and then at 65C for a further 10 min. First strand cDNA was prepared using the Takara RT-PCR kit according to the manufacturers instructions. To verify that RNA was suitable for RT-PCR, amplification was performed using primers based on the conserved sequences of small subunit rRNA. The sequences of small subunit rRNA primers were: SSR1: 5-TTAGACCGAAACCAATGCG-3 SSR2: 5-CGACAACCTAATGCCAGCG-3 Thermocycling conditions were: 95C for 3 min followed by 40 cycles of denaturing at 94C for 30 s, annealing at 55C for 30 s, and extension at 72C for 90 s. After the 40th cycle, there was a final extension step at 72C for 10 min. Reaction products were resolved on 1% agarose gel and visualized under UV light following EtBr staining. Cloning PCR products into T-vectors and sequencing The amplified small subunit rRNA fragment was purified from agarose gels and cloned into the vector PMD18-T (TaKaRa) as described by the manufacturer. The ligation product was used to transform Escherichia coli DH5. Clones carrying recombinant plasmids were collected and sent to Sangon (Shanghai, China) for sequencing.

Results and discussion When applied to L. japonica, three methods previously modified for tissues high in phenolics and polysaccharides failed to yield RNA that was usable for further investigations (Table 1, Figs. 1, 2). Method 1 Method 1 featured isolation and purification of undegraded RNA by treating cells with guanidine thiocyanate-containing lysis buffers without CsCl ultracentrifugation. Chloroform was added to facilitate partitioning of aqueous and organic material. Upon phase separation, RNA remained in the aqueous phase while DNA and proteins were extracted into the protein interface and organic phase. RNA was recovered by precipitation with isopropanol and collected by centrifugation. Seaweed tissues have high levels of polysaccharides and secondary metabolites that are released after disruption of cells embedded in viscous polysaccharides (Ho et al. 1996). Chaotropic salts of guanidinium inhibited RNase, but could be unsuitable for tissues high in polysaccharides or phenolics (Wilkins and Smart 1996). In our experiment, many polysaccharides remained in the RNA sample (Fig. 1), making this method unsuitable for use in L. japonica. The A260/280 and A260/230 ratios were low (Table 1), and the total

236 Table 1 RNA yield and quality by spectrophotometric evaluation Method Absorbance ratiosa A260/230 1 2 3 4
a b

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RNA yield (g.g1 tissue)b A260/280 1.22 1.27 1.57 1.94 (0.08) (0.11) (0.09) (0.05) 25 0.16 12 68

Total time required (h)

0.98 1.38 1.73 2.08

(0.07) (0.07) (0.06) (0.06)

56 1820 11.5 2.53

Expressed as the mean of three samples (standard error) Three samples were combined in one tube; the value given is the combined total yield

RNA using this protocol was unsuitable for RT-PCR amplification (Fig. 2). Method 2 Method 2 used SDS to make cells soluble and inhibit RNase activity (Farrell 1993), and used lower pH to protect RNA from base-catalyzed hydrolysis. PVP can be used for RNA extraction from recalcitrant tissues, and binds and removes phenolic compounds (Wang and Vodkin 1994; Vanessa et al. 2008). EDTA was used in the extraction buffer to bind Mg2+ and Ca2+, which can inhibit RNase activity. Consolidation of the extraction buffer by mixing Tris-HCl-saturated phenol and an acidic solution of sodium acetate caused DNA to be extracted from the aqueous phase and transferred into the interphase and organic phase. RNA could be recovered from the aqueous phase. Differential precipitation with LiCl was used to isolate high molecular weight RNA (Sambrook et al. 1989; Gareth et al. 2006),

and LiCl precipitation at a final concentration of 8 M was carried out to precipitate RNA. Higher LiCl concentration resulted in increased RNA impurity (Hong et al. 1995). This method was modified by using potassium acetate to precipitate polysaccharides into potassium salts in an approach widely used in plant RNA extraction to remove polysaccharides (Ainsworth 1994; Liu et al. 1998; Wang et al. 2008). However, the method failed to extract RNA from L. japonica tissues (Table 1, Fig. 1), which have high levels of phenolic compounds, polysaccharides and some secondary metabolites. Standard methods for extracting RNA from plant tissues employing phenol/SDS were unsuitable because polyphenolic compounds bound to nucleic acids

Fig. 1 Electrophoretic analysis of Laminaria japonica RNA isolated using different extraction methods. Total RNA preparations (35 g) were analyzed on a 1.5% agarose gel stained with ethidium bromide in 1TAE buffer. Three samples for each method from independent extractions using that method were analyzed

Fig. 2 RT-PCR products from total RNA extracted using four methods. RT-PCR products for small subunit rRNA fragments obtained from 1 g total RNA extracted using Methods 1, 2, 3 and 4 were analyzed on a 1.5% agarose gel stained with ethidium bromide in 1TAE buffer. Lanes: M DNA Ladder DL 2,000 (Geneshun, Guangzhou, China), M4M1 methods 41, respectively

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during phenol extraction (Van Driessche et al. 1984; Wu et al. 2002; Wang et al. 2004). The phenol/SDS method is not efficient when applied to tissues containing high levels of secondary metabolites. Method 3 The ready-to-use Trizol reagent isolates total RNA from cells and tissues, and this mono-phasic solution of phenol and guanidine isothiocyanate has been found to be better than the single-step RNA isolation method developed by Chomczynski and Sacchi (1987). During sample homogenization or lysis, Trizol reagent maintains RNA integrity while disrupting cells and dissolving cell components. Adding chloroform/isoamyl alcohol (24:1, v/v) followed by centrifugation separates the solution into an aqueous phase and an organic phase, with RNA present only in the aqueous phase. RNA and DNA were specifically absorbed by the UNIQ-10 column and, after washing with RPE solution, only RNA was left in the column. The simple Trizol reagent method allowed simultaneous processing of many samples and the entire procedure was completed in 1 h. However, the A260/280 ratio and RNA yield was low (Table 1). The 25S and 18S rRNA bands were unclear, with apparent degradation (Fig. 1). Total RNA isolated using this protocol, was unsuitable for RT-PCR amplification (Fig. 2). The Trizol kit did not produce high-quality RNA or acceptable yields with L. japonica because of the presence of alginate and cellulose in the cell walls, and because the tissues contain high levels of phenolic and polysaccharides compounds. The Trizol reagent method is also difficult to use. Sample preparation may have released many secondary metabolites that caused RNA to be lost when those metabolites are eliminated during extraction or precipitation. Method 4 Method 4 was modified from a protocol developed by Apt et al. (1995) for the brown alga Macrocystis pyrifera. To avoid polysaccharide contamination, a high-salt CTAB buffer acted as the cell-disrupting agent and inhibitor of RNase. Most polysaccharides were removed effectively in a single high-salt precipitation at 1.02.5 M NaCl (Fang et al. 1992). The solubility of most proteins lowers at high salt concentrations, and salt seemed to cause co-precipitation of a significant amount of proteinaceous material during the first centrifugation. The presence of DTT at 50 mM avoided oxidative cross-linking of RNA by phenolic compounds and inactivated RNases during homogenization and extraction steps (Gareth et al. 2006). Using ethanol and potassium acetate simultaneously in the extraction buffer aids precipitation of polysaccharides in brown algae (Su and Gibor 1988). A one-tenth volume of 3 M sodium acetate (pH 5.0)

and two volumes of absolute ethanol were used to precipitate RNA selectively, and high-quality RNA was obtained using this protocol. Electrophoresis on an agarose gel showed distinct 28S and 18S rRNA bands (Fig. 1). Total RNA yield was 68 g/g fresh weight (Table 1). The recorded A260/230 and A260/280 were both more than 1.9 (Table 1), suggesting that RNA was effectively separated from protein and polysaccharide. Of the methods tested on L. japonica, this method yielded most RNA of the best quality. The small subunit rRNA fragment (500 bp) was successfully amplified (Fig. 2), and aligning the sequence to GenBank data (Yotsukura et al. 1999) showed a high level of homology (99%) to small subunit rRNA of L. japonica (GenBank accession no. AB022817), confirming RT-PCR product identity. Total RNA isolated by Method 4 was of high integrity, purity and yield, and suitable for downstream molecular research. The principal modifications of the protocol are: (1) the use of toxic chaotropic agents and phenol is avoided; (2) high concentrations of DTT inhibit RNase activity and prevent oxidative cross-linking of nucleic acids by phenolics; (3) ethanol and potassium acetate are used simultaneously to precipitate polysaccharides; (4) using one-tenth volume of 3 M sodium acetate (pH 5.0) and two volumes absolute ethanol precipitates RNA selectively; (5) RNA precipitation is complete in 30 min at 80C. Method 4 is thus a simple, rapid, high-throughput, cost-effective and reproducible procedure for the isolation of RNA from L. japonica.
Acknowledgments The authors gratefully acknowledge NSFC (40618001), the Shandong Agriculture Seed stock Breeding Project for financial assistance, and anonymous reviewers for their critical comments and suggestions to improve the manuscript. Thanks are also kindly given to Mr. Donald Sturge for English revision.

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