Volume: 4, 2025
4th International PhD Student’s Conference at the University of Life Sciences in Lublin, Poland:
ENVIRONMENT – PLANT – ANIMAL – PRODUCT
Abstract number: P007
DOI: https://doi.org/10.24326/ICDSUPL4.P007
Published online: 9 April 2025
ICDSUPL, 4, P007 (2025)
Optimisation of nucleic acid isolation for next generation sequencing
Wiktoria Jędrys1*, Tomasz Ociepa1
1 Institute of Genetics, Plant Breeding and Biotechnology, Department of Agrobioengineering, University of Life Science in Lublin, Akademicka 15, 20-950 Lublin, Poland
* Corresponding author: wiktoria.jedrys@up.lublin.pl
Abstract
The use of modern sequencing techniques in the study of plant genetic material provides much valuable information for a detailed understanding of genetic structure, organisation and regulation. This knowledge translates into breeding plants resistant to biotic and abiotic stresses through selection and creation of plants resistant to diseases and adverse breeding conditions. First-generation sequencing methods require relatively small amounts of genetic material of moderate purity. With the advent of high-throughput next-generation sequencing (NGS), the approach to isolating and preparing DNA/RNA for genomic and transcriptomic analysis has changed significantly. NGS requires high-quality, pure and concentrated genetic material, which poses a major challenge when isolating genetic material from plants, which are particularly rich in polyphenols, polysaccharides and secondary metabolites. The use of commercial isolation kits, despite their convenience and rapid execution, can be expensive and inefficient on a large scale. Therefore, optimising classical methods for the isolation of genetic material from plant tissues may yield better results. In the present experiment, nucleic acid isolation methods were compared for NGS-type sequencing. Material in the form of leaf fragments from oat seedlings was used for the study for DNA, the commercial E.Z.N.A HP Plant DNA Mini Kit (Omega Bio-tek) and the CTAB method were used. For RNA isolat, the E.Z.N.A Plant RNA Kit (Omega Bio-tek) and the TRIzol reagent-based method were used. Both quantitative measurements were made by spectrophotometry (NanoDrop) and fluorometric (Qubit). Finally, integrity was assessed with DIN and RIN parameter calculations on a TapeSation4150 instrument. The study showed that the optimised CTAB method gave better results compared to the minicolumn method. Additionally, the optimised minicolumn-based method gave better results compared to the method using TRIzol. The choice of isolation method and the process itself depend on many factors, such as the type of genetic material to be isolated and the type of sample. Particularly for NGS, it is crucial to meet high quality standards, i.e. the DIN/RIN integrity parameter as high as possible, as even minor contamination or degradation can significantly affect the sequencing results.
Keywords: DNA isolation, RNA isolation, NGS, CTAB method
How to cite
W. Jędrys, T. Ociepa, 2025. Optimisation of nucleic acid isolation for next generation sequencing. In: 4th International PhD Student’s Conference at the University of Life Sciences in Lublin, Poland: Environment – Plant – Animal – Product. https://doi.org/10.24326/ICDSUPL4.P007