Abundant high-quality RNA from medicinal plants for molecular applications

Medicinal plants provide cure for several human and animal diseases. As a pre-requisite towards manipulating gene expression and engineering metabolites, efficient RNA isolation protocol from medicinal plants is needed. In the present study, four recent protocols of RNA isolation were compared based on the electrophoretic pattern as well as, the A260/A280 and A260/230 ratios of RNAs isolated from an important medicinal plant species namely, Rhazya stricta as well as, nine other medicinal plants grown in the Mecca region of the kingdom of Saudi Arabia. The protocols of modified SDS acid phenol and Institute of Himalayan Bio-resource Technology (IHBT) resulted in the most abundant highquality RNAs. Synthesized cDNAs were checked by real time-qPCR and differential display-PCR (DDPCR). The extracted RNAs of the IHBT protocol were proven to be more suitable for downstream molecular applications, especially metabolic engineering.


INTRODUCTION
Medicinal plants represent the basis of the folkloric medicine; the main source of natural products used as pharmaceuticals, agrochemicals, flavor and fragrance ingredients, food additives, and pesticides (Newman et al., 2000;Alyemeni et al., 2010).Biotechnological approaches, specifically recombinant DNA technology and metabolic engineering, have potential as a supplement to traditional agriculture in the industrial production of valuable bioactive metabolites (Ramachandra et al., 2002).Substantially, pure and un-degraded RNA is mandatory for all these applications (Thanh et al., 2009;Takahashi et al., 2010).Large number of protocols for the isolation of RNAs from medicinal plants has been developed and/or modified.However, leaf tissues possess oxidized phenolics and polysaccharides, which bind to RNA and hinder its isolation (Ding et al., 2008;Wang et al., 2008).Commercial kits (ex., TRIzol®, Invitrogen, USA; RNeasy®, Qiagen, Germany, etc) are found to be unsuitable for isolating RNAs from many medicinal plant species (Lal et al., 2001) and results of the present study, especially, those rich in secondary metabolites.The latter often co-precipitate with RNA and also affect its yield and quality (Bugos et al., 1995;Ghawana et al., 2007).In addition, RNA molecules are subject to enzymatic degradation by RNases, which makes RNA unsuitable for first strand cDNA synthesis and real time-PCR (Gasic et al., 2004;Iandolino et al., 2004).Hence, several RNA isolation protocols need to be tested and modified.The protocol of choice should be rapid, simple, economic and devoid of toxic chemicals.In the present study, the most recent RNA isolation protocols were compared for the production of abundant high-quality RNAs from 10 medicinal plant species; some of which were tested for downstream functional genomics analyses, that is, real time-qPCR and DD-PCR.

MATERIALS AND METHODS
A total of 10 medicinal plant leaves were collected from the Mecca region, Saudi Arabia.Some leaves were snap-frozen in liquid nitrogen and stored at -80°C and the rest were dried, preserved, mounted on standard herbarium sheets and identified (Chaudhary and Al-Jowaid, 1999;IPNI, 2008;El-Ghazali et al., 2010).The Mecca region is regarded as a natural reservoir for the collection of

RNA isolation, cDNA synthesis and real time-qPCR
Four recent RNA isolation protocols were used.They are the modified CTAB, modified SDS acid phenol, SDS-LiCl (Hou et al., 2011) and IHTB (Ghawana et al., 2011).Precaution was taken in order to keep eppendorf tubes and tips RNase-free.Other isolation protocol like acid guanidinium thiocyanate-phenol-chloroform-LiCl (Chomczynski and Sacchi, 1987), Invitrogen TRIzol® and Qiagen RNeasy were used for comparison.After extraction, aliquots of extracted total RNAs were stored at -80°C to avoid degradation.RNAs were run on a standard formaldehyde agarose gel (1%) and visualized using UV transilluminator.The quantity and quality of isolated RNAs were assessed visually and by measuring the A260/A280 and A260/A230 ratios.Then, cDNAs from the isolated RNA templates were synthesized using RevertAid TM H M-MuLV reverse transcriptase (Thermo Fisher Scientific, Fermentas, Lithuania).
Real time-PCR was carried out to recover a 101-bp fragment of the actin gene (accession no.X55749), as a house-keeping control, using the Agilent Mx3000P qPCR Systems (Agilent technologies, Palo Alto, CA, USA) with specific primers (forward 5` GCTTCCCGATGGTCAAGTCA 3`; reverse GGATTCCAGCTGCTTCCATTC, Nicot et al., 2005).The reaction components were 12.5 µl Maxima™ SYBR Green/ROX qPCR Master Mix (Thermo Fisher Scientific, Fermentas, Lithuania), 0.2 µM of each forward or reverse primer, and water (nuclease-free water) up to 22.5 µl.Then, 2.5 µl of diluted cDNA template (1/10) was added.Amplification was carried out in triplicates along with a no-template negative control (nuclease-free water).To avoid false positives due to DNA contamination, PCR reaction was carried out for all RNA samples.The thermal cycling conditions consisted of 1 cycle at 95 o C with 2 min for primary denaturation (hot-start), followed by 40 cycles of denaturation at 95°C for 15 s, 57°C for 30 s and extension at 72°C for 30 s. Data were collected and amplification plots of ∆Rn versus cycle number were generated for analysis.

DD-PCR
RNA Image kit and protocol (GenHunter Corporation, Nashville, TN, USA) were utilized to generate cDNA -as template -for DD-PCR with anchor (T11C) and random primers (ARP1).Mixture of PCR reaction (4 µl) and loading dye (2 µl) was heated at 80°C for 2 min, and 2 µl was loaded and run on 6% denatured polyacrylamide gel and visualized by silver staining following manufacturer protocol (SILVER SEQUENCE™ DNA Sequencing System, Promega, Madison, WI, USA).

Drug discovery from traditional medicinal plants continue
Sabir 5215 to provide new and important leads against various illnesses (Hostettmann et al., 2000;Balunas and Kinghorn, 2005;El-Ghazali et al., 2010;Saganuwan, 2010).The use of plants as medicine throughout history is well documented (http://en.wikipedia.org/wiki/Herbalism).Such practices, in turn, might lead to irreversible damages in the fragile ecosystems (Sher et al., 2004).As a result, many economically and pharmaceutically important plant species become rare and sparse in several parts of Saudi Arabia (Earth, 2003;Sher and Hussain, 2009).Such endangered plant species ought to be mined for functioning genes for abiotic stress resistance and/or pharmaceutical applications as an alternative avenue to preserve their genomes.
Additionally, applying biotechnological approaches like metabolic engineering and pathway optimization are in the main focus of academia and industry to reduce costs and increase metabolite productivity (Keasling, 2010).The latter approaches can also improve nutritional and health promoting effects of nutraceuticals and reduce the amount of unwanted by-products with potential toxic or allergic activities.Such a progress relies on the possibility to study and manipulate patterns of medicinal plant gene expressions, which emphasizes the need to get highly purified RNA samples.

RNA yields and quality for different isolation protocols
Several standard protocols for RNA isolation including acid guanidinium thiocyanate-phenol-chloroform-LiCl, Invitrogen TRIzol® and Qiagen RNeasy were applied for isolating RNA, but they all failed to yield RNAs from medicinal plants (data not shown).The main reason was the presence of phenolic compounds, polysaccharides and complex secondary products (Schultz et al., 1994).The four recent protocols for RNA isolation under study (modified CTAB, modified SDS acid phenol, SDS-LiCl and IHBT protocol) were compared for the quality and yields of resulted RNAs isolated from 10 medicinal plants in the Mecca region, KSA.The success of an RNA isolation protocol may be judged by the quantity, purity, and integrity of RNA recovered (Wang et al., 2011).Electrophoretic analysis (running on a denaturing gel) showed that the two RNA bands of the 10 plant species were vague, less sharp and highly degraded when isolated by the modified CTAB protocol (data not shown).The results for the other three protocols indicated that the quality and yields of RNAs per 100 mg leaf tissue for most plant species isolated by either IHBT and modified SDS acid phenol protocols are higher than those isolated by SDS-LiCl protocols (Figures 1a to c and 2a-c and Table 1).
The Institute of Himalayan Bioresource Technology (IHBT) protocol resulted in more consistent RNA yields (78.8 to 91.4 µg) as compared to the other two protocols (10.9 to 95.1 µg for SDS acid phenol and 24.9 to 59.7 µg for SDS-LiCl protocol).Spectrophotometric measurements were made at OD 230, 260 and 280.The first indicates the level of polysaccharides contamination (Loulakakis et al., 1996); the second indicates the level of nucleic acids, while the third indicates the level of protein contamination (Winfrey et al., 1997).The A260/280 and A260/230 ratios were high (1.85 to 1.98 and 2.19 to 2.61, respectively) for IHBT protocol, while low for SDS acid phenol (1.71 to 2.00 and 1.89 to 2.20, respectively) or SDS-LiCl (1.59 to 1.74 and 1.47 to 1.90, respectively) protocols.These results reflect that the highest levels of protein and polysaccharides contaminations across the 10 plant species are shown in the SDS-LiCl followed by SDS acid phenol protocols.
The IHBT protocol (Ghawana et al., 2011) is guanidinium salt-free, phenol-based.Presence of guanidinium salt was known to promote dissociation of RNA from non-protein complex that would further inhibit RNA isolation (Ding et al., 2008;Wang et al., 2008;Ghansal et al., 2009).Phenol functions as a strong protein denaturant and inhibitor of RNase; SDS and EDTA are also inhibitors of RNase.Further, pH of the solution was maintained in the acidic range to allow efficient and preferable partitioning of RNA in the aqueous phase leaving DNA in the phenolic phase (Wallace, 1987).An appropriate concentration of NaOAc was also included in the solution to aid precipitation of RNA in the presence of isopropanol.The addition of DEPC-treated autoclaved water rendered sufficient aqueous environment for partitioning of RNA into the aqueous phase.

Downstream molecular analysis of RNAs of selected plant species
Different RNA preparations from four selected plant species (that is, Rhazya stricta, Catharanthus roseus, Zygophyllum simplex, Calotropis procera), with the best characteristics, were further tested in triplicates for quality using real time-qPCR.The reactions for RNAs isolated by IHBT and modified SDS acid phenol protocols resulted in low C t (Threshold cycle) values (18.88 to 16.72 and 17.94 to 23.44, respectively, Table 2), while high for SDS-LiCl (28.14 to 39.71).The results of agarose gel electrophoresis (Figure 3) were confirmatory in which one-band pattern of the actin gene fragment (101 bp) was shown for the reactions with the four plant species for the IHBT and modified SDS acid phenol protocols, while only R. stricta for SDS-LiCl protocol.
The dissociation curve for the first two protocols reflected the previous finding, besides, resulted in no mismatches or primer dimers (Figure 4), while the SDS-LiCl gave no consistent amplification due to the high C t value.Although the three protocols could produce abundant high-quality RNA, only two were amenable to real time-qPCR.Digestion of RNA with RNase-free   DNase (Ghawana et al., 2010;Xu et al., 2010) resulted in no DNA contamination in which direct PCR for RNA samples gave negative results (data not shown).DD-PCR analysis for the four selected plant species utilizing the best two RNA isolation protocols indicated that IHBT resulted in the production of high-as well as low-molecular-weight cDNAs, while the modified SDS acid phenol resulted in the production of low-molecularweight cDNAs, only (Figure 5).The latter amplicons are well-known to be non-useful for subsequent molecular  analysis and harbor no gene fragments.Thus, homology searching with these short sequences will likely recover no hits in the gene bank.Accordingly, it can be concluded that IHBT protocol is the ideal for RNA isolation of the tested plant species to be utilized for downstream molecular analysis and metabolic engineering of medicinal

Figure 1 .
Figure 1.Leaf RNA yields and quality for various plant species (1-10, see Materials and Methods) as assessed by denaturing agarose gel electrophoresis isolated by the IHBT (a), SDS-LiCl (b) and Modified SDS acid phenol (c) protocols.

Table 1 .
Yields and other spectrophotometric measurements for RNA isolated from leaves of the 10 plant species (1-10) using three protocols.

Table 2 .
Comparison of Ct values for various RNA isolation protocols from leaves of four selected plant species (1, 2, 5 and 6 as assessed by real time-qPCR.