Extraction of functional total RNA from Cayratia trifolia.
RNA isolation is the initial step involved in the study of gene expression and also in the utilization of genes for genetic improvement. Any RNA isolation protocol must succeed in extracting RNA species from cellular and extra-cellular components. Therefore, it is crucial to eliminate DNA, protein, polysaccharides, phenolic compounds and other secondary metabolite compounds during the RNA extraction process. Inability to obtain clean, intact RNA may result in the failure of downstream applications such as cDNA synthesis, RT-PCR, cDNA display and cDNA synthesis.
The extraction of RNA from plant tissues can be complicated and most of the time requires modification of existing protocols or development of novel procedures. In this study, we used Cayratia trifolia as a plant material. C. trifolia plant was selected based on preliminary data gathered by our group which showed that the plant is capable of accumulating high amount of heavy metals and hydrocarbon. Our long term research objective is to isolate genes which confer plant resistance to heavy metals and hydrocarbons. C.trifolia is well known for their diversity in secondary metabolite, polysaccharide, and polyphenolic compounds [1,15]. All of these components would co-precipitate with the RNA and constitute the major obstacle of RNA isolation and also often hinder RNA preparation or compromise RNA sample quality [6,10]. Many of the established RNA isolation protocols are based on highly toxic chaotropic agents such as phenol, phenol/chloroform or guanidine thiocyanate which quickly denature the endogenous ribonucleases in tissues . However, these methods often failed to significantly eliminate carbohydrate and phenolic compounds resulting in a poor RNA yield.
We developed a method to isolate good quality RNA from leaves of C. trifolia after significant modification of the method from RNeasy Plant Mini Kit established by Qiagen. High molecular weight polyethylene glycol (PEG 8000) was added in the extraction buffer (RLC buffer) to bind to the phenolic compounds which were then eliminated by ethanol precipitation . We proved that the isolated RNA is of high quality
and quantity by optical density and agarose gel analyses. Results from RT-PCR experiment confirmed that RNA was functional.
Materials and methods
C. trifolia plants were collected from Petronas Penapisan Melaka Sdn. Bhd. (PPMSB), Melaka, Malaysia. The plants were grown in the Universiti Kebangsaan Malaysia's greenhouse under normal soil and petroleum sludge. Fresh leaves were collected and labeled according to the habitat (sludge and normal soil). Leaves were then cleaned with sterile distilled water and used for RNA extraction.
Isolation of RNA:
The protocol was as described by the RNeasy Plant Mini Kit (QIAGEN) instruction manual. All the equipments and solutions used in the protocol were treated with diethylpyrocarbonate (DEPC) as described by Sambrook et al. . PEG 8000 was added to the extraction buffer (RLC buffer) to a final concentration of 2%. P-mercaptoethanol was added into RLT and RLC buffers with the ratio of 10 [micro]L [beta]ME per 1 mL buffer and properly mixed. Four volumes of 100% undenatured ethanol were added into RPE buffer.
Determination of RNA quality and Quantity:
The quality and quantity of RNA were analyzed by measuring optical density (OD) using Biophotometer (Eppendorf AG, Germany). RNA quantity was determined by diluting 1 [micro]l of RNA sample into 49 [micro]l of sterile DEPC-treated water and OD was measured at 260nm wavelength. RNA quality was determined by measuring OD at 230 nm, 260 nm and 280 nm. The rationings of [OD.sub.260]: [OD.sub.230] and [OD.sub.260]: [OD.sub.280] were then calculated. The quantity and quality of RNA were also determined by electrophoresis on 2.0 % (w / v) agarose gel. The gel was stained with ethidium bromide  and visualized using Vilber Lourmat INFINITY-CAP gel imager (Germany).
A good quality total RNA was used for the first strand cDNA synthesis using SuperScript III First-Strand Synthesis System for RT-PCR (Invitrogen, USA) according to the manufacturer recommendations. Oligo(dT) primer was used as a primer for cDNA synthesis by SuperScript III Reverse Transcriptase. A total of 3[micro]g RNA was used as a template.
Second Strand cDNA synthesis and Differential cDNA display:
Differential cDNA display was performed using GeneFishingTM DEG Premix Kits (Seegene) according to the instruction manual provided. Fifty ng of the first-strand cDNA was used as a template for the second strand cDNA synthesis. The reaction mixture consisted of 5 [micro]M of one of the 20 arbitrary ACP primers (ACP1-ACP20) (Seegene), 10 [micro]M dTACP2 and [SeeAmp.sup.TM] [ACP.sup.TM] Master Mix. The mixture was incubated at 94[degrees]C for 5 minutes and 50[degrees]C for 3 minutes for the second strand cDNA synthesis reaction to take place. Final incubation at 72[degrees]C for 1 minute was required for the reaction to complete. The mixture was then subjected to PCR for 40 cycles at 94[degrees]C for 40 second, 65[degrees]C for 40 second and 72[degrees]C for 40 second followed by final incubation at 72[degrees]C for 5 minutes. The PCR products were separated on 2% (w / v) agarose gel and stained with ethidium bromide as recommended .
Results and discussion
A commercially available RNeasy Plant Mini Kit for RNA extraction was tested for its efficiency to extract total RNA from the leaves of C. trifolia. The kit was supplied with two lyses buffer, i.e. RLC and RLT. Both buffers were tested in addition to a slight but significant modification by addition on PEG 8000 to a final concentration of 2%. Figure 1 shows the total RNA samples obtained from the procedure. RNA extraction from leaves of C. trifolia grown in normal soil and sludge using unmodified RLT buffer produced a very poor and unusable RNA (CT1 and CT1). Addition of PEG 8000 to RLT buffer slightly improved yield of RNA from both C. trifolia leaves (CTP1 and CTP1).
Change of lyses buffer from RLT to the RLC buffer did not produce any RNA product (CC1 and CC2). However, addition of PEG 8000 to the RLC buffer produced significant results. RNA obtained from both C. trifolia leaves indicated the presence of 2 clear and distinct bands for 25S and 18S rRNA (CCP1 and CCP2). C. trifolia leaf grown on normal soil gave a slightly lower amount of RNA compared to the C. trifolia leaf grown in sludge. As a comparison, tobacco was used as well. RNA extraction using unmodified RLT buffer showed the presence of 2 clear and distinct bands proved that the RNeasy Plant Mini Kit was a very efficient system for RNA extraction.
A summary of spectrophotometry analysis of RNA samples is shown in Table 1. RNA samples obtained from tobacco and C. trifolia leaves extracted with the presence of PEG 8000 were of a good quality as indicated by the ratio for both [OD.sub.260 / 280] and [OD.sub.260 / 230] of more than 1.8. RNA extractions from C. trifolia leaves without the use of PEG 8000 produced a significantly very low RNA concentration which was not detected on agarose gel (Figure 1).
The RNeasy Plant Mini Kit provides a choice of lyses buffers, i.e. RLT and RLC, which contain guanidine thiocyanate and guanidine hydrochloride respectively. In most cases, RLT buffer is the lyses buffer of choice due to the greater cell disruption characteristic and denaturation properties of guanidine thiocyanate. Many established works used RLT for RNA extraction from leaves and gave good quality of RNA [9,13,14]. However, due to the amount and the type of secondary metabolites presence in C. trifolia tissue [1,15], the use of guanidine thiocyanate is not recommended as it can cause solidification of the sample, making extraction of RNA impossible.
[FIGURE 1 OMITTED]
Different protocols often produce different quality of RNA preparation. Tow[??] et al.  found that a better preparation of RNA was obtain by applying phenol:chloroform during extraction procedure. Dineen et al.  had tested 5 different types of commercial kits to extract RNA from virus and all of them produced different qualities of RNA preparation. Djami-Tchatchou and Straker,  used cetyltrimethylammonium bromide-based protocol without using phenol to extract RNA from skin and flesh of avocado fruit. Gehrig et al.  found that the use of PEG 8000 during RNA extraction greatly improved the yield and served as a better template for reverse transcription and PCR amplification. PEG 8000 is a reducing agent that eliminates the polysaccharides and prevents the oxidation of phenolic compounds. Phenolic and other compounds that may interfere with RNA were bound to PEG 8000 in extraction buffer and subsequently eliminated by precipitation as an initial step to RNA extraction [2,7]. In cells, total RNA consists mainly of mRNA, tRNA and rRNA which constitute approximately 3%, 17% and 80% respectively. During extraction, the ratio of RNA recovery is also followed by the ratio of different types of RNA in cells. Therefore, a good quality of RNA preparation is often indicated by the presence of clear 25S and 18S rRNA bands. Our results (Figure 1) again proved that the RNA recovery and yield were greatly improved by the addition of PEG 8000 into the lyses buffer.
RNA obtained from these modified method served as a robust template for reverse transcription as indicated by PCR amplification. Figure 2 shows results of cDNA display from C. trifolia grown in sludge compared to C. trifolia grown in normal soil. PCR products analysed on 2% (w / v) agarose gel showed the presence of differentially display cDNA bands. Twenty cDNA bands were either qualitatively or quantitatively displayed in C. trifolia grown in sludge. Meanwhile 7 cDNA bands were either qualitatively or quantitatively displayed in C. trifolia grown in normal soil.
The presence of differently display cDNA bands indicated that genes representing the particular cDNA were differently expressed in that particular sample compared to others. Petroleum sludge contains high concentration of toxic components such as heavy metals  and hydrocarbons . In our case, differently expressed genes in C. trifolia grown in sludge may represent genes involved in plant resistance mechanism or genes expressed due to plant stress against toxic compounds of either heavy metals or hydrocarbons that presence in the sludge. These results proved that the total RNA obtained from the extraction protocol was of high quality. In the future, we will clone and identify these cDNAs. These cDNAs may represent genes of interest particularly the one that could be used in bioremediation.
[FIGURE 2 OMITTED]
We have successfully isolated total RNA of a good quality from C. trifolia by modification of method established by Qiagen. cDNA display experiments showed the presence of display bands probed that the RNA was functional.
The authors are grateful to the Universiti Kebangsaan Malaysia and Petronas Research Sdn. Bhd. (PRSB). This work was fully funded by grants STGL-010-2008 from PRSB.
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Roslina Mat Yazid and Nik Marzuki Sidik
School of Biosciences and Biotechnology, Faculty of Science and Technology Universiti Kebangsaan Malaysia, 43600 Bangi, Malaysia
Roslina Mat Yazid and Nik Marzuki Sidik: Extraction of functional total RNA from Cayratia trifolia
Nik Marzuki Sidik, School of Biosciences and Biotechnology Faculty of Science and Technology 43600 Universiti Kebangsaan Malaysia Bangi, Malaysia
E-mail: email@example.com; Tel: +603 89215998; Fax: +603 89252698
Table 1: RNA quality measured by UV spectrophotometry. Leaf samples Type of [OD.sub.260/280] lysis buffer used Tobacco--TB RLT 2.00 C. trifolia RLT 1.54 (normal soil)--CT1 C. trifolia RLT 1.65 (sludge)--CT2 C. trifolia (normal RLT + PEG 8000 1.98 soil)--CTP1 C. trifolia RLT + PEG 8000 1.80 (sludge)--CTP2 C. trifolia (normal RLC 1.61 soil)--CC1 C. trifolia RLC 1.58 (sludge)--CC2 C. trifolia (normal RLC + PEG 8000 1.94 soil)--CCP1 C. trifolia RLC + PEG 8000 1.98 (sludge)--CCP2 Leaf samples [OD.sub.260/230] RNA conc. ([micro]g/ [micro]L) Tobacco--TB 1.90 109.15 C. trifolia 0.469 3.04 (normal soil)--CT1 C. trifolia 0.45 2.08 (sludge)--CT2 C. trifolia (normal 1.85 37.87 soil)--CTP1 C. trifolia 1.82 44.18 (sludge)--CTP2 C. trifolia (normal 0.50 4.00 soil)--CC1 C. trifolia 0.64 5.60 (sludge)--CC2 C. trifolia (normal 1.85 64.80 soil)--CCP1 C. trifolia 1.90 86.84 (sludge)--CCP2
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|Title Annotation:||Original Article|
|Author:||Yazid, Roslina Mat; Sidik, Nik Marzuki|
|Publication:||Advances in Environmental Biology|
|Date:||Sep 1, 2011|
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