Page 71 - Book of Abstracts

5 International Scientific Online Conference DOI: https://doi.org/10.15414/2021.9788055224015

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CHOICE OF METHOD FOR RNA EXTRACTION FROM TISSUES OF FOUR PLANT SPECIES
RICH IN BIOLOGICALLY ACTIVE SUBSTANCES
Alena Kabachevskaya, Grazhyna Niescier
Institute of Biophysics and Cell Engineering of the National Academy of Sciences of Belarus,
Minsk, Belarus; E-mail.: kabachevskaya@lab.ibp.org.by
Tissues of medicinal and aromatic plants are characterized by a high content of various
secondary metabolites, which often interfere with the RNA extraction from plant cells.
Meanwhile, high-quality RNA is the basis of molecular biological, genetic, physiological, etc.
plant research. There are a lot of protocols for RNA isolation from plant tissues, but most of
them involve the use of volatile toxic substances, which makes these methods not very practical
for everyday usage. This work aimed to select effective methods for the RNA extraction from
leaves of some plant species rich in biologically active substances, as well as to reduce the use
of toxic reagents. Both well-known medicinal and aromatic species (Echinacea purpurea (L.)
Moench, Ocimum basilicum L.) and the representatives of traditional medicine with
antiproliferative, antitumoral, or cytotoxic potential (Plectranthus amboinicus (Lour.) Spreng.,
Plectranthus scutellarioides Blume) were investigated. The following methods of RNA
extraction were used: 1) with Tri-reagent (according to the Sigma protocol); 2) 2 % CTAB (cetyl
trimethyl ammonium bromide, 1.4 M NaCl, 20 mM EDTA, 100 mM Tris-HCl, 2 %
polyvinylpyrrolidone (PVP)), isopropanol 100 %, DTT (DL-dithiothreitol), SDS (sodium
dodecyl sulfate), 70 % ethanol, DNase. 3) 100 mM Tris-HCl, 20 mM EDTA, 2 M NaCl, 2 % CTAB,
1 % PVP, 2 % 2-mercaptoethanol, 24:1 chloroform: isoamyl alcohol, LiCl, 80 % ethanol. 4)
Modified method No. 3, in which 2-mercaptoethanol, chloroform, and isoamyl alcohol are
replaced by dithiothreitol and 1-bromo-3-chloropropane, that allows reducing the volume of
volatile toxic substances used in the work.
For each investigated plant species an optimal RNA extraction method was revealed: for
basil, the trizol method turned out to be the most optimal, for Echinacea – method No. 2, for the
most difficult to isolate RNA plant material, Plectranthus spp. – methods 3 and 4, and it should
be noted that since method 4 involves the use of fewer volatile toxic substances, it is preferable
for use in work. Method No. 2 is also characterized by reduced use of solvents, however, the
yield and purity of RNA from plants of the genus Plectranthus are not high.
So, the high content of biologically active substances in medicinal and aromatic plants, as
well as the use of highly toxic reagents when working with these species, is a key problem in
the study and analysis of gene expression at the earliest stages of work. The methods described
in our work represent a solution to eliminate these problems and make it possible to carry out
further larger-scale experiments.

Keywords: Echinacea purpurea, Plectranthus amboinicus, Plectranthus scutellarioides, Ocimum
basilicum, RNA extraction.
Acknowledgments
We are grateful for providing useful information on method 3 of RNA extraction from Plectrantus
amboinicus to Prof. Madya Dr. Janna Ong Binti Abdullah, Faculty of Biotechnology & Biomolecular
Sciences UPM and Mohd Hairul Ab Rahim, Lecturer Faculty of Industrial Sciences & Technology,
Universiti Malaysia Pahang, Malaysia.

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5 International Scientific Conference Agrobiodiversity for Improving the Nutrition, Health, Quality of Life and |70
Spiritual Human Development
November 3 2021
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