ANALYTICALBIOCHEMISTRY

Analytical Biochemistry 328 (2004) 90–92

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Notes & Tips

Isolation of high-quality nucleic acids from Cistus creticus ssp. creticus and other medicinal plants�

Irene Pateraki and Angelos K. Kanellis¤

Group of Biotechnology of Pharmaceutical Plants, Laboratory of Pharmacognosy, Department of Pharmaceutical Sciences,Aristotle University of Thessaloniki, 541 24 Thessaloniki, Greece

Received 11 November 2003

Leaves of Cistus creticus ssp. creticus (Cistaceae) EDTA, 1.5% (w/v) sodium dodecyl sulfate, 1% (w/v)

secrete several labdane-type diterpenes, which displayeither cytotoxic activity against a number of human leuke-mic cell lines or antibacterial and antifungal activity [1,2].Considering the characteristics and possible future appli-cations of these metabolites it seemed worthwhile to studytheir biosynthesis and metabolism at the molecular level.Thus, it was essential to establish methods for nucleic acidextraction and puriWcation from C. creticus tissues.

Cistus species contain high amounts of metabolites thatinterfer with nucleic acid isolation, such as terpenoids,polyphenols, Xavonoids, Xavonoid aglycones, and glyco-sides, resin, and thick waxy cuticle covering the aerialparts’ epidermis and especially the leaves [3,4]. As a conse-quence of these characteristics, several published methodsor kits for nucleic acid isolation (e.g., Nucleospin RNAplant kit; Macherey–Nagel, Duren, Germany) [5–10] thatwere applied to this plant had unsatisfactory results. Theonly method that gave good-quality total RNA was theone described in Loulakakis et al. [8] but the yield was rel-atively low (data not shown). In this report, we describeoptimized protocols that yield large amounts of high-quality genomic DNA and total RNA from C. creticus tis-sues. The described methods were appropriate for nucleicacid isolation independently of the sampling period, plantage, or plant cultivation method and worked eYciently forother medicinal plant species also.

The total RNA extraction procedure is as follows:I Grind 1 g of leaf tissue in Wne powder with mortarl

and pestle in liquid nitrogen and place it in a sterile50-ml tube with 20 ml prechilled extraction buVer[200 mM Tris–HCl, pH 8.5, 300 mM LiCl, 10 mM

� This research was supported by a grant from the Greek GeneralSecretariat of Research and Technology (PENED 99ED 637) awardedto A.K.K.

¤ Corresponding author. Fax: +30-2310-997662.E-mail address: kanellis@pharm.auth.gr (A.K. Kanellis).

0003-2697/$ - see front matter 2004 Elsevier Inc. All rights reserved.doi:10.1016/j.ab.2004.01.030

sodium deoxycholate, 1% (v/v) Igepal CA 630 (FlukaChemie, Gmbh., Buchs, Switzerland), 5 mM thiouria,10 mM dithiothreitol, 1 mM aurintricarboxylic acid].Add 6 ml of 20% polyvinylpyrrolidone (PVP)1 justbefore homogenization.

I Homogenize the solution with Polytron PT 2100(Kinematica, Switzerland) at full speed for approx.30 s. Centrifuge the homogenate at 10,000g for 15 minat 4 °C to remove insoluble particulates.

I Filter the supernatant through sterilized miracloth(Calbiochem, La Jolla, CA, USA) into an Oak Ridge-type tube.

I Precipitate nucleic acids with 1 volume isopropanoland 0.1 volume of 3 M sodium acetate. Incubate at¡20 °C for at least 1 h. Centrifuge the solution at10,000g for 20 min and resuspend the pellet in 6 mlextraction buVer.

I Extract the sample with equal volume of nonequili-brated phenol/chloroform. Centrifuge the mixture at10,000g for 10 min at 4 °C and rescue the upper phase.

I Add CTAB and NaCl at Wnal concentrations of 1%(w/v) and 0.7 M, respectively. Incubate the mixture at65 °C for 15 min and extract the solution with equalvolume of chloroform, for carbohydrate removal.

I Centrifuge the mixture at 10,000g for 10 min at 4 °Cand rescue the upper aqueous phase.

I Add 10 M LiCl to a Wnal concentration of 3 M forselective RNA precipitation. After at least 2 h incuba-tion at ¡20 °C centrifuge the sample at 10,000g for30 min.

I Resuspend the pellet in 500 �l diethyl pyrocarbonate(DEPC)-treated water and perform a Wnal precipitation

1 Abbreviations used: PVP, polyvinylpyorolidone; DEPC, diethylpyrocarbonate; FW, fresh weight; CTAB, cetyltrimethylammoniumbromide.

Notes & Tips / Analytical Biochemistry 328 (2004) 90–92 91

step with 2 volumes of absolute ethanol and 0.1 vol-ume of 3 M NaAC, pH 5.2, with at least 2 h incuba-tion at ¡20 °C.

I Spin down the RNA solution for 30 min at 4 °C,resuspend the pellet in 100 �l DEPC-treated water,and store at ¡80 °C.For genomic DNA extraction a similar procedure

with slight diVerences was followed. The RNA extrac-tion buVer was used in a diVerent ratio [10 ml buVer pergram of fresh weight (FW) tissue with 3 ml 20% PVP]without aurintricarboxylic acid. Cell disruption wasachieved using heat treatment (65 °C for 1–2 h), becausemechanical cell disruption (homogenization) resulted inslightly sheared DNA (data not shown). Heat-treatedsamples yielded approximately 10 times more DNA thannon-heat-treated samples (data not shown). Isopropanolprecipitation was performed at room temperature toreduce the carryover salts and the pellet was resuspendedin 5 ml TE buVer (10 mM Tris–HCl, pH 8.0, and 1 mMEDTA, pH 8.0) prior to the phenol/chloroform step.Tris–HCl pH 8.0, equilibrated phenol was used for DNAisolation instead of nonequilibrated phenol, and the LiClstep was replaced with a second isopropanol precipita-tion for eYcient DNA isolation.

All glass- and plasticware were autoclaved for 30 minat 125 °C prior to use, while solutions for RNA extrac-tion were treated with 0.1% DEPC for at least 3 h andthen autoclaved; 20% PVP solution was prepared by dis-solving the PVP K30 (MW 40,000) powder in DEPC-treated water. Nonequilibrated phenol/chloroformreagent was prepared by mixing 500 g of crystal phenolwith 150 ml DEPC-treated water, 70 ml m-Cresol, and0.5 g 8-hydroxyquinoline, supplemented with equal vol-ume of chloroform. Tris–HCl, pH 8.0, equilibrated phe-nol was prepared according to Sambrook et al. [11].

A critical step for successful RNA preparation wasthe ratio of extraction buVer volume to C. creticus FWtissue. Ten milliliters buVer per gram of FW tissue sup-plemented with 3% PVP gave satisfactory results, withregard to total RNA quality and quantity, for growth-chamber-cultivated plants. For Weld-grown plants, it wasnecessary to use 20 ml extraction buVer and 6% PVP for1 g of FW tissue to obtain good-quality RNA. ThesediVerences are probably due to variation in polyphenolcontent in these samples [4].

Addition of PVP and not polyvinylpolypyrrolidone inthe extraction buVer during the cell lysis procedure wasimportant as this resulted in 25% higher nucleic acidyields (data not shown). Soluble PVP was subsequentlyremoved from the solution during the chloroformextraction step. The detergents contained in the extrac-tion buVer proved to be of great signiWcance, because nonucleic acids were isolated when extraction buVers withno detergents (or lower concentrations of them) wereused (data not shown). They helped in more eYcient celllysis (synergistically with heat treatment for DNA and

mechanical homogenization for RNA) as they dissociatecell membranes. Moreover they were probably responsi-ble for the breakdown of complexes formed betweennucleic acids and insoluble cell particles that are precipi-tated in the Wrst step of most protocols used duringwhich cell debris were discarded.

Another advantage is the omission of the ultracentrif-ugation step described in Loulakakis et al. [8]. Protein andpolysaccharide removal were achieved by phenol/chloro-form and CTAB/chloroform extractions, respectively.

The presence of discrete ribosomal RNA bands(Fig. 1A) suggests that RNA degradation was negligible.Further, these RNA preparations were devoid of chro-mosomal DNA contamination. The method provedeYcient for total RNA extracted from Cistus creticusssp. creticus, Myrtus communis, Lavandula hybrida, andEbenus cretica leaves (Fig. 1A). Yield was satisfactoryfor all species, ranging from 118 to 194�g/g FW. Theratio A260/A280 was from 1.82 to 1.85, indicating thepurity of the samples. Further, the quality of total RNAfrom C. creticus was tested by the successful applicationof RT-PCR (Fig. 1C) and RNA blot analysis (data notshown). The single sharp band, which was observed afterRNA blot hybridization, indicated the absence of degra-dation (data not shown). For RT-PCR reactions cDNAwas synthesized from C. creticus young leaves totalRNA and oligo(dT) primer (50-ACTAGTCTCGAG(T)19-30). Synthesized cDNA was used as template forCcGGPPS1 and CcGGPPS2 (C. creticus geranylgeranyldiphosphate synthase gene 1 and 2, respectively) geneampliWcation (Fig. 1C) using gene-speciWc primers

Fig. 1. Electrophoretic and RNA blot analysis of total RNA fromdiVerent plant species. (A) 5 �g of total RNA extracted from Cistuscreticus ssp. creticus (lane 1), Lavandula hybrida (lane 2), Myrtus com-munis (lane 3), and Ebenus cretica (lane 4), and from C. creticus roots(lane 5), Xower buds (lane 6), stems (lane 7), and leaves (lane 8) ana-lyzed in 1% agarose gel stained with ethidium bromide. (B) Total RNAextracted from young leaves (lanes 1 and 3) and glandular trichomes(lanes 2 and 4) of C. creticus visualized in agarose gel with ethidiumbromide (lanes 1 and 2), or blotted and hybridized with radiolabeledCcGGPPS1 (lanes 3 and 4). (C) RT-PCR products for CcGGPPS 30

ampliWcation (lane 1, molecular weight markers; lane 2, CcGGPPS1;lane 3, CcGGPPS2) resulted from cDNA synthesized from total RNAisolated from C. creticus young leaves.

92 Notes & Tips / Analytical Biochemistry 328 (2004) 90–92

(GGPPS1.FOR 50-GTTGGCGTATCCCCCGCCCG-30

and GGPPS2.FOR 50-TGGGGGCCTCGCCGGATAA-30) and oligo(dT). The protocol developed here wasused to isolate total RNA from C. creticus glandulartrichomes, roots, Xower buds, and stems with excellentresults (Figs. 1A and B).

Genomic DNA extraction from leaves of C. creticusssp. creticus, Salvia oYcinalis, M. communis, L. hybrida,and E. cretica yielded 238, 127, 93, 173, and 14�g DNAper gram of tissue FW, respectively. All samples wereintact with no shearing and exhibited good gel migration(Fig. 2A). The yield was estimated by measuring theabsorbance at 260 nm after RNase treatment while theA260/A280 ratio, ranging from 1.81 § 0.09 to 1.94 § 0.06,indicated the purity of the preparations. DNA from allsamples was eVectively subjected to complete digestionswith HindIII restriction endonuclease (Fig. 2A) and toPCR ampliWcation (Fig. 2B). For PCRs, genomic DNAextracted from all plants mentioned above was used astemplate in diVerent concentrations, from 5 to 100 ng,with equal eYciency (data not shown), implying thequality and purity of the extracts. 18S ribosomal DNAwas selected for ampliWcation, because it is highlyconserved among all plant species, using the 18S.FOR50-CTGGTTGATCCTGCCAGT-30 and 18S.REV 50-ATTACCGCGGNTGCTGGC-30 primers. GenomicDNA from C. creticus digested by several restriction

Fig. 2. Electrophoretic and Southern blot analysis of genomic DNAsamples. (A) Genomic DNA extracted from Cistus creticus ssp. creticus(lanes 2 and 8), Salvia oYcinalis (lanes 3 and 9), Lavandula hybrida(lanes 4 and 10), Myrtus communis (lanes 5 and 11), and Ebenus cretica(lanes 6 and 12) and analyzed in 1% agarose gel. In lanes 8–12 genomicDNA has been subjected to HindIII restriction endonuclease digestion.The 1-kb DNA ladder (Gibco BRL, Life Technologies) was used asmolecular weight marker (lane 1). (B) PCR ampliWcation of a 560-bp18S rDNA fragment using as template 25 ng genomic DNA extractedfrom C. creticus (lane 2), S. oYcinalis (lane 3), L. hybrida (lane 4), M.communis (lane 5), and E. cretica (lane 6). The 100-bp DNA ladder(Invitrogen) was used as molecular weight marker (lane 1). (C) South-ern blot of C. creticus genomic DNA (10 �g/lane) digested with EcoRI(lane 1), AvaI (lane 2), NdeI (lane 3), EcoRI–AvaI (lane 4), EcoRI–NdeI(lane 5), and AvaI–NdeI (lane 6) and hybridized with radiolabeledCcGGPPS1. Molecular weights (in bp) are indicated at the left.

endonucleases was used for Southern blot analysis ofCcGGPPS1 gene, giving sharp and distinct hybridiza-tion signals (Fig. 2C).

Consequently, the methods described here for totalRNA and genomic DNA isolation can be used forextraction of superior-quality nucleic acids free of con-taminants. They were applied eVectively to more thanone plant species rich in secondary metabolites, indepen-dently of plant cultivation method or plant age. Nucleicacids isolated according to these methods were biologi-cally active and were used successfully in several enzy-matic reactions. Total RNA isolated was used eVectivelyfor cDNA synthesis, RT-PCRs, and RNA blot analysiswhile genomic DNA could be ampliWed by PCR andcompletely digested with restriction endonucleases.

Acknowledgment

We thank Dr M. Kalamaki for reading the manuscript.

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