Extraction of High-Quality RNA from Rubber Tree Leaves

Liu-Hong DENG,1 Ming-Wu LUO,2;y Chun-Fa ZHANG,1 and Hui-Cai ZENG1

1Institute of Tropical Bioscience and Biotechnology, Chinese Academy of Tropical Agricultural Sciences,Haikou 571101, China2College of Materials Science and Chemical Engineering, Hainan University, Haikou 570228, China

Received January 13, 2012; Accepted April 21, 2012; Online Publication, July 7, 2012

[doi:10.1271/bbb.120014]

A specific technique capable of producing high-quality RNA for rapid amplification of cDNA ends(RACE) was established for challenging tissues: leavesof the rubber tree. Total RNA was extracted bycetyltrimethylammonium bromide (CTAB)-LiCl com-bined with TRIzol reagent. The isolated RNA was highlyintact. With RNA as template, full-length cDNA wasobtained (NCBI, AY461413) by RACE.

Key words: rubber-tree leaves; RNA extraction; cetyl-trimethylammonium bromide (CTAB);TRIzol; rapid amplification of cDNA ends(RACE)

Although more than 2000 plant species have beenfound to possess the ability to synthesize natural rubber,the rubber tree is still the only one that qualified asa commercial source for natural rubber production.Rubber tree leaves challenge commonly used RNAextraction techniques in that they contain not onlyrubber (specific secondary metabolites), but also largeamounts of polysaccharides, pigments, polyphenol com-pounds and rigid cell walls. Sodium dodecyl sulfate(SDS) is used to harvest RNA from rubber-tree leavesand latex.1,2) In our study, however, we found that RNAprepared by SDS-based methods from rubber-treeleaves, although suitable for common RT-PCR andNorthern blot, was unsuitable for RACE in terms ofintegrity, since RACE performance makes greaterdemands on the integrity of RNA than common RT-PCR does. Other techniques, including cetyltrimethy-lammonium bromide (CTAB)3–6) and TRIzol,7,8) did notwork well for rubber-tree leaves either. Thus the presentstudy was motivated by a need to develop a usefultechnique capable of producing high-quality RNA fromleaves of the rubber tree in particular for use in RACE.

Rubber trees grown at the Chinese Academy ofTropical Agricultural Science, Hainan, were harvestedto extract RNA from leaves of the adult plant. One g ofsamples was snap-frozen in liquid N2 and stored at�80 �C until use. The frozen leaf tissues were ground toa fine powder. The powder was suspended in 10mL ofpre-warmed extraction buffer containing 2% (w/v)CTAB, 100mmol�L�1 Tris–HCl (pH 8.0), 100mmol�L�1 EDTA, 1.4mol�L�1 NaCl and 5% (v/v) �-mercaptoethanol, and the suspension was incubated at

65 �C for 30min. Then 10mL chloroform/isoamylalcohol (24:1, v/v) was added to the suspension, andwas centrifuged at 12;000� g for 10min at 4 �C. Thesupernatant was transferred to a fresh tube, and 1/3 volof 8mol�L�1 LiCl was added, and mixed well, andstored at �20 �C overnight. The reaction mixture wascentrifuged at 12;000� g for 20min at 4 �C to produce arude RNA pellet. The rude pellet was subsequentlydissolved in 1mL of TRIzol buffer, and then mixed with0.2mL of chloroform, and centrifuged at 12;000� gat 4 �C for 15min. The aqueous phase was transferredto another new tube, to which 0.5mL of isopropylethanol was added, and was stored at �20 �C for 30min.Then the mixture was centrifuged at 12;000� g for20min at 4 �C. The final pellet was washed with 70%(v/v) ethanol and air-dried. The integrity and purity ofthe isolated RNA was examined by 1.2% formaldehydeagarose gel electrophoresis at an absorbance ratio ofA260/A280.This new extraction technique was compared with

conventional extraction methods including SDS, CTAB,guanidinium thiocyanate (GT),9) and TRIzol by assess-ing the resulting RNA yield, purity (A260/A280), andintegrity (28S/18S). The new technique gave the highestRNA yield and the A260/A280 value of the isolatedRNA was in the desired range, 1.8–2.0. This indicatesthat the RNA prepared by the technique was of highpurity and free from polyphenol, polysaccharide, andprotein contamination (Table 1). In contrast, the othermethods gave poor A260/A280 values (all below 1.8),which indicates that the RNA was contaminated byprotein and other organic compounds (Table 1). Theratio of 28S/18S of the RNA prepared by the newmethod was close to the ideal value of 2.0, withoutshowing any significant downward shift on a form-aldehyde agarose gel (Fig. 1A), indicating that theintegrity of the RNA was good. In contrast, the othermethods gave 28S/18S values of far less than 2.0, andshowed significant downward shifts on a formaldehydeagarose gel (Fig. 1C), indicating that severe RNAdegradation occurred during the extraction process.Moreover, both the TRIzol and the GT reagent producedbrown RNA pellets, while CTAB and SDS producedRNA pellets insoluble in water. Possible reasons are asfollows: When rubber-tree leaves are ground to apowder, high levels of polyphenols in the leaf tissues

y To whom correspondence should be addressed. Tel: +86-898-66962819; Fax: +86-898-36686779; E-mail: luomw@163.comAbbreviations: CTAB, cetyltrimethylammonium bromide; GT, guanidinium thiocyanate; PCR, polymerase chain reaction; RACE, rapid

amplification of cDNA ends; RT, reverse transcription; SDS, sodium dodecyl sulfate

Biosci. Biotechnol. Biochem., 76 (7), 1394–1396, 2012

Note

are readily oxidized to form covalent compounds withRNA, which leads to RNA oxidation and degradation.Rubber and polysaccharides in leaf tissues may also bindRNA and thereby make it more difficult to isolate andpurify the RNA. The new approach can solve theseproblems by the novel combination of CTAB-LiCl andTRIzol. Since initial CTAB-LiCl treatment effectivelyseparated RNA from rubber, polyphenol and polysac-charide contamination and prevented the oxidation ofpoly-phenols under alkaline conditions, which avoidedproducing brown pellets.10) LiCl precipitation, althoughtime-consuming, was necessary, probably because thepolysaccharide concentration was still high. SubsequentTRIzol treatment removed the protein contaminationdeeply, and thus forestalled the production of insolublepellets.

To test the stability of the RNA, a small portion wasincubated at 37 �C for 2 h, and then compared with aduplicate control stored at �70 �C. The 28S/18S ratio ofthe sample incubated at 37 �C was almost the same asthat of the control (Fig. 1B), indicating the RNAcontained no nuclease contaminants.

The purity and integrity of RNA is the key factor forsuccessful RACE. To further validate the quality of theresulting RNA, we performed RACE with the isolatedRNA as template. Briefly, the missing 50 and 30 ends of acDNA fragment was obtained by 50- and 30-RACE,following the manufacturer’s instructions (SMARTer�RACE cDNA Amplification Kit; Clontech, MountainView, CA, USA). F1 (50-TGGCATTCACCACCATGT-CACCT-30) was as 50-RACE primer and R1 (50-CCTACCATGCTCAATTGCGTCTCCA-30) as 30-RACE.According to the sequence information obtained in 50-and 30-RACE production, the full-length cDNA was

successfully amplified by RT-PCR (Fig. 2). The com-plete cDNA was 1,365 bp long, containing a 1,149-bpORF encoding Hevea acyl-CoA reductase with atheoretical molecular weight of 43.5 kDa and an iso-electric point of 8.97, which was available from theNCBI database under accession no. AY461413.In summary, the new technique described here can

effectively produce high-quality RNA from rubber-treeleaves. It overcomes the limitations of the convention-ally used SDS-based method by using a new combina-tion of well-established methods. It involves (i) initialCTAB-LiCl treatment; (ii) further TRIzol treatment. Theresulting RNA was sufficiently intact to allow successfulamplification of full-length cDNA in RACE.

Acknowledgments

This project supported by the National NaturalScience Foundation of China (Grant no. 31101200),and by the Hainan Provincial Natural Science Founda-tion of China (Grant no. 310082).

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A B C

Fig. 1. Total RNA of H. brasiliensis on 1.2% Formaldehyde Agarose Gel.A, RNA prepared by the present method. 1, RNA of latex; 2, RNA of leaves. B, RNA stored for 2 h. 1, RNA stored at �70 �C; 2, RNA stored

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Fig. 2. 30- and 50-RACE and RT-PCR Amplification of Hevea Acyl-CoA Reductase Gene.

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Table 1. Yield and Quality of Total RNA Prepared by Five DifferentMethods

Extraction method A260/A280RNA yield

(mg�g�1 of fresh weight)

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CTAB 1:7� 0:1 202� 11:2

SDS 1:6� 0:2 124� 10:3GT 1:5� 0:3 80� 13:1

TRIzol 1:5� 0:1 75� 10:7

Results are expressed as mean� SD (n ¼ 3).

Extraction of High-Quality RNA from Rubber Tree Leaves 1395

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