ICDSUPL2-P018

Volume: 2, 2023
2^nd International PhD Student’s Conference at the University of Life Sciences in Lublin, Poland:
ENVIRONMENT – PLANT – ANIMAL – PRODUCT

Abstract number: P018

DOI: https://doi.org/10.24326/ICDSUPL2.P018

Published online: 19 April 2023

ICDSUPL, 2, P018 (2023)

Validation of genomic RNA extraction methodology from the leaves of sugar beet (Beta vulgaris L. subsp. vulgaris)

Karolina Różaniecka¹*, Michał Nowak¹, Krzysztof Kowalczyk¹

¹ Institute of Plant Genetics, Breeding and Biotechnology, University of Life Sciences in Lublin, Akademicka 15, 20-950 Lublin, Poland

^{* Corresponding author: karolina.rozaniecka@up.lublin.pl}

Abstract

Ribonucleic acid is unstable and sensitive to many factors, therefore methodology of its extraction from tissue must provide conditions that protect it from degradation. Obtaining material with the best possible qualitative and quantitative parameters is crucial for confident results, especially for quantitative methods like quantitative PCR (qPCR) and digital PCR (dPCR). The most important quality parameters for RNA samples are integrity, concentration, and purity. Sugar beet is one of the most important root crops grown in Poland, being the main raw material for sugar production. Due to its high content of sugars and secondary metabolites, sugar beet tissue is one of the more challenging plant materials for RNA isolation. The working hypothesis assumes that genomic RNA isolation from sugar beet by means of different methodologies will result in significant differences in the quality of the samples obtained. The aim of the study was to validate the methodology of genomic RNA isolation from sugar beet leaf samples using one reagent and four ready-to-use reagent kits. In the presented study leaf samples collected from the field conditions from four genotypes of sugar beet plants were analyzed. The harvested plant material was immediately frozen in liquid nitrogen and placed at -80°C. In the next step material was subjected to RNA isolation using the method based on the TRIzol reagent (Invitrogen) as well as four kits from different manufacturers, according to recommended protocols. The integrity of isolated RNA samples was analyzed by means of separation on a 2% agarose gel and capillary electrophoresis using TapeStation 4150 system (Agilent Technologies). The concentration was measured using the Qubit fluorometer (Invitrogen) and purity was determined by means of the NanoDrop spectrophotometer (Thermo Scientific). The use of different isolation methods resulted in RNA samples with varying parameters. The integrity of all obtained samples was good, while the amount and purity of the obtained material differed significantly. The best qualitative and quantitative results were obtained using a E.Z.N.A.® Plant RNA Kit (Omega Bio-Tek) based on RNA purification on a silica bed on minicolumns. Obtained results proved that any experiment requiring RNA extraction should be preceded by validation and optimization of its methodology, since some of the standard protocols do not allow obtaining samples of a quality that ensures the reliability of subsequent results.

How to cite

K. Różaniecka, M. Nowak, K. Kowalczyk, 2023. Validation of genomic RNA extraction methodology from the leaves of sugar beet (Beta vulgaris L. subsp. vulgaris). In: 2nd International PhD Student’s Conference at the University of Life Sciences in Lublin, Poland: Environment – Plant – Animal – Product. https://doi.org/10.24326/ICDSUPL2.P018