Plant Physiology and Biochemistry 47 (2009) 1113–1115

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Plant Physiology and Biochemistry

journal homepage: www.elsevier.com/locate/plaphy

Short Communication

Isolation of good quality RNA from a medicinal plant seabuckthorn,

rich in secondary metabolites
Rajesh Ghangal a, Saurabh Raghuvanshi b, Prakash Chand Sharma a, *
a
University School of Biotechnology, Guru Gobind Singh Indraprastha University, Kashmere Gate, Delhi 110403, India
b
Department of Plant Molecular Biology, University of Delhi South Campus, New Delhi 110021, India

a r t i c l e i n f o a b s t r a c t

Article history: Medicinal plants are being widely investigated owing to their ability to produce molecules of therapeutic
Received 21 April 2009 significance. Isolation of good quality RNA is a tedious but primary step towards undertaking molecular
Accepted 11 September 2009 biology experiments. However, medicinal plants are rich in secondary metabolites and not amenable to
Available online 17 September 2009
standard RNA isolation protocols involving Guanidine isothiocyanate (GITC). So an RNA isolation protocol
from difficult samples (richer in secondary metabolites) is of highest desiderata. Here we propose a new
Keywords:
protocol suitable for isolating RNA from plant tissues rich in secondary metabolites. To standard CTAB
CTAB
(Cetyl Trimethyl Ammonium Bromide) buffer, addition of 2% PVPP (polyvinyl polypyrrolidone) and
Medicinal plants
RNA isolation 350 mM b-mercaptoethanol was found useful. Use of glacial acetic acid (1M) along with ethanol for
Seabuckthorn precipitation after phenolization and chloroform extraction enhanced the RNA yield. This is the first report
Secondary metabolites of using glacial acetic acid in a CTAB based protocol for the precipitation of RNA. This protocol has been
validated in medicinal plant Hippophae rhamnoides vern. seabuckthorn, where standard RNA isolation
methods involving GITC and TRIZol extraction buffers failed. The RNA isolated by this method was of good
quality as gauged by spectrophotometric readings and denaturing agarose gel electrophoresis. To the best
of our knowledge, this RNA isolation protocol has never been published before. The RNA thus obtained
could be suitably used for the downstream molecular procedures like Reverse Transcription Polymerase
Chain Reaction (RT-PCR), Real Time-PCR, cDNA library construction, etc.
Ó 2009 Elsevier Masson SAS. All rights reserved.

1. Introduction to unveil the synthesis of the bioactive substances in seabuckthorn at

Medicinal plants have drawn attention of present day researchers
as they produce important therapeutic molecules. The medicinal
properties of these plants can be attributed to the presence of high
amounts of polysaccharides, polyphenols, hydrozable tannins and
other secondary metabolites. During the past decade, the seabuck-
thorn (Hippophae sp.) has attracted immense global attention
because of its multifold medicinal value. The reference to medicinal
use of seabuckthorn has been found in the ancient Greek texts of
Theophrastus and in classic Tibetan medicinal texts including ‘‘the
RGyud Bzi’’ (The four books of Pharmacopoeia) dating back to the
times of Tang Dynasty (618–907AD) [1]. Application of molecular
biology tools may help in better exploitation and conservation of
seabuckthorn genetic resources. Further, it would also be interesting
Abbreviations: b M.E, b-mercaptoethanol; CTAB, Cetyl Trimethyl Ammonium
Bromide; GITC, Guanidine Isothiocyanate; PVP, polyvinyl pyrrolidone; PVPP, poly-
vinyl polypyrrolidone.
* Corresponding author. Tel.: þ91 11 23900220; fax: þ91 11 23900221.
E-mail address: prof.pcsharma@gmail.com (P. Chand Sharma).
the molecular level.
Seabuckthorn can withstand extremes of temperature from 55  C
to 43  C, soil pH from 5.8 to 9.5 and grows well under drought
conditions of about 250–800 mm annual rainfall. It harbors
a symbiotic nodule forming mycorrhizal fungus, Frankia that fixes
atmospheric nitrogen [2]. In China, the plant has been used to
replenish the eroded lands and control soil erosion [3]. Therefore,
this plant is a very good candidate for studying the molecular
mechanisms involved in abiotic stress tolerance. Monitoring alter-
ations in gene expression in response to environmental changes
provides necessary inputs in understanding molecular basis of stress
tolerance. Availability of good quality RNA is the primary require-
ment in undertaking these investigations. However, extracting RNA
from seabuckthorn tissue is a difficult task as it is known to be rich
in polysaccharides, polyphenols and secondary metabolites [1,4,5].
Phenolic compounds are known to get readily oxidized to form
quinones which in turn bind to nucleic acids and hinder isolation of
good quality RNA [6]. Here, we present a new protocol for RNA
isolation from plant tissues with high phenolic contents, combining
the use of CTAB with varying concentration of other chemicals and

0981-9428/$ – see front matter Ó 2009 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.plaphy.2009.09.004
1114 R. Ghangal et al. / Plant Physiology and Biochemistry 47 (2009) 1113–1115
precipitation using glacial acetic acid. To the best of our knowledge,
this protocol has not been described earlier.
2. Results and discussion
2.1. RNA isolation methodology
There are many published RNA isolation protocols, of which, single
step method by Guanidine Isothiocyanate (GITC) [7] is the most
widely used and has proved useful in model plants i.e., rice and
Arabidopsis. As most of the RNA isolation protocols use GITC based
extraction buffer, we proceeded with guanidine based method in
seabuckthorn, which did not yield any RNA. PVP or PVPP are known to
form complex with polyphenolics through hydrogen bonding and
thus help in the removal of polyphenolics from the homogenate.
In our case, using PVP/PVPP during extraction also did not prove
helpful. Even increased GITC concentration in the extraction buffer
did not help. Commercially available extraction kits and solutions (TRI
reagent, Sigma) also gave poor results. Commercial kits produced
dark brown pellet which was difficult to re-suspend. During every
isolation protocol, we took wheat leaves as control and wheat RNA
showed bright and sharp bands at the end of each of the protocols
(Fig. 1a–c). The failure of guanidine based methods may be explained
by the presence of high levels of secondary metabolites in seabuck-
thorn leaves and RNA was readily lost together with these complexes
during subsequent removal of the precipitate. Guanidinium salts are
protein denaturants and are not effective agents for dissociating RNA
from non-protein complexes like polyphenolics [8].
When guanidine based method repeatedly gave negative results,
we switched over to CTAB based method, and appropriately
modified the protocol as per the requirements of RNA isolation from
a phenolic rich plant tissue. The improved CTAB protocol described
here efficiently eliminated most of the interfering molecules and
yielded translucent and water soluble RNA pellets. CTAB buffer, as
described by Chang et al. [9] was used for this purpose. CTAB was
used as a cell disrupting agent, supplemented by insoluble PVPP
instead of soluble PVP as it is incompatible with phenol extraction
and would have obstructed RNA precipitation [10]. PVPP as an
alternative is insoluble, cross-linked form of PVP with high
molecular weight which is chemically inert and forms complex
with polyphenols through hydrogen bonds, allowing them to be
Fig. 1. Agarose gel profile of total RNA using different isolation methods a. Total RNA
from wheat leaves using GITC based method b. Total RNA from wheat leaves using
GITC-PVP based method c. Total RNA from wheat leaves using GITC-PVPP based
method d. Total RNA from seabuckthorn leaves using our modified CTAB method.
separated from nucleic acids [11]. The concentration of b-mercap-
toethanol was increased in extraction buffer to avoid polyphenol
oxidation as higher concentration helps to preserve RNA by pre-
venting production of quinones from phenolics. It also inactivates
RNases by breaking the disulphide linkages [12]. Increasing the
volume of the extraction buffer to 5 times with respect to leaf tissue
seemed helpful as it diluted polyphenolics [13]. Phenol is usually
used for removing proteins and at initial stages ensures inhibition
of RNase activity, but has detrimental influence on downstream
applications of RNA such as RT-PCR, cDNA library construction, etc.
Therefore, Chloroform/Isoamyl alcohol (24/1) was used to remove
residual phenol and proteins.
RNA is not shear sensitive as they are not bulky molecules but it is
very labile to alkali as it has been speculated that RNA is more sensitive
to strand cleavage by OH ions at higher pH [14,15]. Therefore, the
phenol used was water saturated (pH 5.0) and 1 M Glacial acetic acid
kept the pH low. This is the first report of using glacial acetic acid for
precipitation of RNA in a CTAB based RNA isolation protocol. After
precipitating, RNA pellet was washed with 5 M NaCl to remove
polysaccharides [16].
2.2. Electrophoresis of RNA
RNA thus obtained was spectrophotometricaly quantified and
examined for the possible contamination of protein. The yield of
total RNA obtained was 600–700 mg/g of lyophilized leaf tissue,
with A260/A280 ratio being 1.70. The integrity of RNA was assessed
by the brightness and sharpness of rRNA bands visualized on
denaturing 1.2% agarose gel (containing 1.1% formaldehyde). The
RNA sample and RNA premix [10 MOPS/Formaldehyde/Form-
amide (1/3.5/10)] were mixed in 3/1 ratio. Heat shock was given to
remove any secondary structure by keeping the above sample
mixture at 65  C for 5 min and immediately plunging into ice for
5 min. This sample along with loading dye was run at 5 V/cm in
MOPS running buffer. Distinct bands were visualized under UV
transilluminator and ratio of brightness between 28 S rRNA and
18 S rRNA was nearly 2/1 and there was no sign of RNA degradation
(Fig. 1d).
2.3. cDNA library construction
Since reverse transcription is highly sensitive to impurities, it was
necessary to obtain pure RNA for successful construction of full
length cDNA libraries. cDNA population of the range 0.5–4 kb was
selected for preparing cDNA library. We successfully constructed
cDNA library from seabuckthorn leaf RNA isolated using our modi-
fied protocol. To check the percentage of insert in cDNA library,
plasmids were isolated using alkaline lysis method and digested
with SmaI and XbaI. The digested products were run on 1% agarose
gel and nearly each clone had an insert (Fig. 2).
Fig. 2. Agarose gel profile of restriction digested recombinant plasmids (Lanes 2 to 15)
along 1 kb ladder (Lane 1).
 R. Ghangal et al. / Plant Physiology and Biochemistry 47 (2009) 1113–1115
3. Material and methods
3.1. Plant material
Seabuckthorn leaves were plucked and snap frozen in liquid
nitrogen from the seabuckthorn plantation in DIHAR (Defence
Institute of High Altitude Research), Leh, India. The frozen tissue
was transported in liquid nitrogen and stored at 80  C on reaching
the laboratory, until use.
3.2. RNA isolation protocol
In a pre-chilled mortar & pestle,1 g of freeze-dried leaf tissue
material was finely ground using liquid nitrogen, and quickly
transferred in 5 ml of pre-warmed CTAB extraction buffer [2% w/v
CTAB, 100 mM Tris–Cl (pH 8.0), 2 M NaCl, 25 mM EDTA (pH 8.0)]
containing 100 mg PVPP and 150 ml b-mercaptoethanol. The
mixture was shaken vigorously for 1 min, and then incubated at
65  C in water-bath for 20 min with intermittent mixing. Cooled
down the mixture to room temperature and then added equal
volume of Chloroform/Isoamyl alcohol (24/1) and mixed well by
invert mixing. Centrifuged the contents at 10,000  g for 25 min at
4  C. To carefully collected upper aqueous phase, equal volume of
Phenol (pH 5.0)/Chloroform/Isoamyl alcohol (25/24/1) was added
and mixed well, followed by centrifugation at 10,000  g for 15 min
at 4  C. Carefully collected upper aqueous phase (without disturb-
ing the interface) and added equal volume of Chloroform/Isoamyl
alcohol (24/1), with the repeat of centrifugation step again. To the
upper aqueous phase, 0.2 volume of 1 M Glacial acetic acid, 0.1
volume of 3 M sodium acetate and 2 volume of ethanol were added
(mixed by tapping). The pellet was collected by centrifugation after
incubating the tubes at 80  C for 2 h. Washed the pellet twice with
5 M NaCl first and once by 70% ethanol by re-suspending the pellet
and centrifuging at 12,000  g for 15 min at 4  C at each of the wash
step. Allowed ethanol to evaporate and re-suspended the pellet in
minimum amount of Diethyl Pyrocarbonate (DEPC) treated water.
3.3. cDNA library construction
mRNA was purified from 300 mg of total RNA using Oligotex mRNA
purification kit (Qiagen, Canada). cDNA amplification was performed
by Long Distance PCR (LD-PCR) as specified in SMART cDNA Library
Construction kit (Clontech, Japan). SfiI digested double stranded
cDNA was ligated into linearised pDNR-Lib vector (SfiI digested).
Ligated product was electroporated in electrocompetent Escherichia
coli DH5a cells. Transformation mixture was plated onto LB-chlor-
amphenicol plates and incubated at 37  C overnight. Glycerol stocks
of transformed colonies were prepared and stored at 80  C.
4. Conclusion
The proposed protocol proved efficient and suitable for isolation
of good quality RNA from seabuckthorn leaves, a medicinal plant
which contains large amounts of secondary metabolites, polyphenols
1115
and hydrozable tannins in tissues. The modified CTAB buffer con-
taining greater concentration of PVPP proved successful, as it effi-
ciently removed polyphenols in the very first step of extraction. Such
a cellular population of RNA molecules, free of polyphenols, when
precipitated using 1 M glacial acetic acid in combination with ethanol
kept the pH of solution low, which in turn ensured recovery of pure
RNA in larger quantities. RNA obtained by this protocol was of good
quality as indicated by spectrophotometry and amenable to down-
stream molecular applications. The new protocol is likely to find
application in RNA isolation from other medicinal plant species or
tissues which are extraordinarily rich in secondary metabolites. The
protocol is also likely to prove equally efficient with the fresh and
freeze-dried tissues.
Acknowledgements
We thank DIHAR (Defence Institute of High Altitude Research),
Leh, India for providing seabuckthorn samples. This work was
supported by grants from DRDO (Defence Research and Develop-
ment Organisation), India and research fellowship from ICMR
(Indian Council of Medical Research), India.
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