A Rapid and Economical Method for Low Molecular Weight RNA Isolationfrom a Wide Variety of Plant Species

Zewei AN,1;2 Yachao LI,1 Lili XIE,1 Qilin ZHAI,1 and Huasun HUANG2;y

1College of Agriculture, Hainan University, Haikou, Hainan 570228, China2State Center for Rubber Breeding and Rubber Research Institute, Chinese Academy of Tropical Agricultural Sciences,Danzhou, Hainan 571737, China

Received March 8, 2013; Accepted April 1, 2013; Online Publication, July 7, 2013

[doi:10.1271/bbb.130187]

We have developed a time- and cost-effective methodfor isolating low molecular weight (LMW) RNA fromplants. In our protocol, the isolation procedure can becompleted within 3 h. Polyethylene glycol (PEG) andabsolute ethanol are used to isolate LMW RNA, and theLMWRNA yields were >80�g/g of fresh-weight tissuesfor several of the plant species tested.

Key words: low molecular weight RNA; microRNA;polyethylene glycol; plant

Low molecular weight (LMW) RNA, consisting of20–24 nt small RNAs, are used in miRNA studies,including cloning, library construction, and expressionanalysis. MiRNAs are non-coding small RNAs inanimals, plants, and viruses.1–3) Since the first miRNAwas reported for Caenorhabditis elegans,4) more than25,141 mature miRNAs have been identified in 193species (http://www.mirbase.org/, Release 19, 2012).

In plants, miRNAs play important biological roles indevelopment,5,6) biotic and abiotic stress responses,7,8)

and metabolic pathways.4,9) Plant miRNAs can beidentified by direct cloning from small RNA libraries10)

or by bioinformatic prediction from existing genomicdatabases, which are based on conserved sequences ofplant miRNAs as well as the ability of the surroundingregion to adopt a fold-back structure.11)

Several methods have been reported for the isolationof plant LMW RNA, such as commercial kits (e.g.,Ambion; Stratagene), traditional methods,12) and theLMW RNA enriched method,13,14) but all of theseextraction protocols are either expensive or time-consuming. Here we propose a revised protocol:isolation of LMW RNA using polyethylene glycol(PEG). PEG can fractionate DNA of different molecularmasses,15) and it has been used to isolate DNA andRNA.16–18) It is an improvement on the Ambioncommercial kit. In the Ambion method, a glass-fiberfilter is used to isolate LMW RNA in the presence ofvarious concentrations of ethanol.

Our revised method consists of cell lysis withcetyltrimethylammonium bromide (CTAB) buffer,enrichment of LMW RNA, and recovery of LMWRNA. The operating steps are as follows: i) Samplepreparation. Grind 0.5 g of leaves to a fine powder with a

mortar in liquid nitrogen. ii) Plant cell lysis. Transfer thepowder to a clear 15-mL polypropylene centrifuge tubeand add 5mL of extraction buffer (2% CTAB, 25mM

EDTA, 2M NaCl, 4% polyvinylpyrrolidone-40 (w/v),100mM Tris–HCl pH 8.0, and 0.5 g/L spermidine) pre-warmed to 65 �C and 200 mL of �-mercaptoethanol tothe tube. Mix by vigorously inverting the tube, and let itrest for 5min at room temperature. iii) Organicextraction (1). Add an equal volume of acid-saturatedphenol and vortex vigorously for 1min. Centrifugeat 12,000 rpm for 15min at 4 �C, and transfer thesupernatant to a fresh 15-mL centrifuge tube. iv)Organic extraction (2). Add an equal volume ofchloroform:isoamyl alcohol (24:1) and vortex vigorouslyfor 1min. Centrifuge at 12,000 rpm for 10min at 4 �C,and transfer the supernatant to a fresh 15-mL centrifugetube. v) Organic extraction (3). Repeat step 3 and 4once, respectively. vi) Purification of LMW RNA (1).Add 1/4 volume of 30% PEG8000 and chill on ice for30min. Centrifuge at 12,000 rpm for 10min at 4 �C. vii)Purification of LMW RNA (2). Transfer the supernatantto a clear 15-mL centrifuge tube, and add 1/3 volume ofabsolute ethanol. Mix by inverting the tube andcentrifuge at 12,000 rpm for 5min at 4 �C. viii)Recovery of LMW RNA. Transfer the supernatant to aclear 15-mL centrifuge tube, and add 1/10 volume ofNaAC and 2/3 volume of absolute ethanol. Mix byinverting the tube and incubate at �20 �C for 30min.Centrifuge at 12,000 rpm for 10min at 4 �C. ix) Wash-ing. Wash the pellets with 70% ethanol twice. x)Dissolution and store. Briefly dry and resuspend thepellets in 80 mL of DEPC-treated water. Store them at�80 �C.LMW RNA was extracted from leaves of Hevea

brasiliensis Muell Arg. (the rubber tree), Arabidopsisthaliana L., Manihot esculenta Crantz (cassava), Musasapientum L. (banana), and Pinus densiflora Sieb. etZucc. (the pine tree). The quality of LMW RNA wasdetermined by running the samples on 1.2% form-aldehyde-agarose gel and 15% urea-PAGE. Our resultsindicated that the LMW RNA bands were clear andsharp in denatured agarose gel (Fig. 1a). Two distinct5.8S and 5S rRNA band, and intense tRNA and miRNAssmears were resolved from LMW RNA on PAGE(Fig. 1b), which indicated that the LMW RNA was

y To whom correspondence should be addressed. Tel: +86-898-2330-0573; Fax: +86-898-2330-0315; E-mail: azwcn@126.comAbbreviations: CTAB, cetyltrimethylammonium bromide; LMW, low molecular weight; PAGE, polyacrylamide gel electrophoresis; PEG,

polyethylene glycol

Biosci. Biotechnol. Biochem., 77 (7), 1599–1601, 2013

Note

intact and efficiently isolated. The purity and concen-tration of the LMW RNA was assessed with aspectrophotometer (Ultrospec 3300 pro, GE Healthcare,Cambridge, England) at wavelengths of 230, 260, 280,and 320 nm. The value of A260=280 and A260=230 rangedfrom 1.80 to 2.02 and from 1.99 to 2.33 (Table 1),respectively, which indicates that there was littlecontamination of the LMW RNA. The yields of LMWRNA isolated by our method were higher than those bypreviously reported methods (Table 2).

To evaluate further the application of LMW RNAisolated by our protocol, we detected HbmiR16619) fromLMW RNA isolated from the rubber tree by the poly(A)-tailed RT-PCR method.20) cDNA amplification wascarried out over 35 cycles at an annealing temperature of60 �C using primer PCR-166 (5-TCGGACCAGGC-TTCATTCCCC-3) and primer PCR-R (5-AAGCAGT-GGTATCAACGCAGAGTAC-3). The expected PCRproducts (78 bp) were detected on 4% agarose gel(Fig. 1c) and were verified by sequencing (Fig. 1d). Ourresults revealed that LMW RNA obtained from therubber tree contained miRNAs and can be used inmiRNAs studies.

Our method of isolating LMW RNA from polysac-charide- and polyphenolic-rich plants is cost-effectiveas compared to commercial kits (e.g., Ambion andStratagene), and is also time-effective as compared tothe methods reported by Carra et al.13) and Wang et al.14)

Our procedure can be completed within 3 h, whereastheir methods13,14) take 29 h and 5 h, respectively. Theregents used in our method are not expensive, and

the extraction buffer used can also be used to isolatehigh-molecular-weight RNA and DNA, as previouslydescribed.18) In our previous study, we developed amethod for the isolation LMW RNA from Arabidop-sis,21) which can work very well in Arabidopsis, but ifyou want use it in other plant species, you must adjustthe concentration of sodium acetate in low salt concen-tration extraction buffer. The procedure described hereis applicable to a wide variety of plant species, includingArabidopsis, pine, cassava, banana, and the rubber tree.The high quality LMW RNA obtained by our procedureis suitable for downstream applications such as smallRNA library construction, deep sequencing, RT-PCR tostudy gene expression, and Northern blotting.

Acknowledgments

This work was partly supported by the NationalNatural Science Foundation of China (30960319,31271796), the Fundamental Research Funds of theRubber Research Institute, CATAS (1630022011002),and the Earmarked Fund for the Modern Agro-IndustryTechnology Research System (CARS-34).

a b c

d

Fig. 1. LMW RNA Quality Isolated by Our Method.a, Electrophoresis pattern of LMW RNA isolated from various plants in 1.2% denature agarose gel. b, Detection of LMW RNA isolated from

various plants in 15% denaturing PAGE. c, RT-PCR detection of HbmiR-166 from LMW RNA isolated from H. brasiliensis: Lane 1 is blankcontrol. In order to detect contamination and no-specific amplification, milli-Q water is used in place of template DNA in blank control. Lane 2to 4 are replicate samples. Lane M is a 20-bp ladder marker (Takara, Dalian, China). d, Sequencing map of HbmiR-166.

Table 1. Quality of the LMW RNA Isolated from Various Materials

Materials A260=280� A260=230

�

H. brasiliensis Muell Arg. 2:00� 0:01 2:05� 0:03A. thaliana L. 1:84� 0:04 2:00� 0:01

M. esculenta Crantz 1:95� 0:03 2:08� 0:06

M. sapientum. L. 2:01� 0:01 2:25� 0:02

P. densiflora Sieb. et Zucc. 1:98� 0:02 2:30� 0:03

�Mean� SEM for three independent samples.

Table 2. Yields of LMW RNA Isolated by Three Different Methods

Methods Materials Yield (mg/g)a,b

Our revised protocol H. brasiliensis Muell Arg. 95:4� 9:6A. thaliana L. 87:3� 9:3

M. esculenta Crantz 86:8� 13:4

M. sapientum L. 99:1� 13:1

P. densiflora Sieb. et Zucc. 94:0� 10:7Wang et al. (2010) Pyrus sorotina Will 12:5� 0:50

Carra et al. (2007) Vitis vinifera L. 62:08� 14:82

A. thaliana L. 57:80� 6:45

Zea mais L. 36:40� 0:11Triticum aestivum L. 45:30� 7:51

Solanum tuberosum L. 28:80� 0:79

aMean� SEM for three independent samples.bYields were determined spectrophotometrically and are presented as mgRNA/g fresh weight.

1600 Z. AN et al.

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