A universal protocol for high-quality DNA and RNA isolation from diverse plant species.

Autor: Masoomi-Aladizgeh F; School of Natural Sciences, Macquarie University, Sydney, NSW, Australia., Jabbari L; Department of Tissue Culture and Gene Transformation, Agricultural Biotechnology Research Institute of Iran (ABRII), AREEO, Karaj, Iran., Khayam Nekouei R; Department of Agronomy and Plant Breeding, Faculty of Agricultural Sciences, University of Guilan, Rasht, Iran., Aalami A; Department of Agronomy and Plant Breeding, Faculty of Agricultural Sciences, University of Guilan, Rasht, Iran., Atwell BJ; School of Natural Sciences, Macquarie University, Sydney, NSW, Australia., Haynes PA; School of Natural Sciences, Macquarie University, Sydney, NSW, Australia.
Jazyk: angličtina
Zdroj: PloS one [PLoS One] 2023 Dec 14; Vol. 18 (12), pp. e0295852. Date of Electronic Publication: 2023 Dec 14 (Print Publication: 2023).
DOI: 10.1371/journal.pone.0295852
Abstrakt: Next-generation sequencing demands high-quality nucleic acid, yet isolating DNA and RNA is often challenging, particularly from plant tissues. Despite advances in developing various kits and reagents, these products are tailored to isolation of nucleic acid from model plant tissues. Here we introduce a universal lysis buffer to separate nucleic acid from various plant species, including recalcitrant plants, to facilitate molecular analyses, such as quantitative PCR (qPCR), transcriptomics, and whole-genome sequencing (WGS). The protocol is a modification of the original CTAB methods, which leads to nucleic acid isolation from many plant species, including monocots and eudicots. The lysis buffer consists of hexadecyltrimethylammonium bromide (CTAB), sodium chloride (NaCl), Tris base, ethylenediaminetetraacetic acid (EDTA) and β-mercaptoethanol (βME). The modified CTAB method enables the isolation of nucleic acid from small amounts of plant tissues (e.g., 15-100 mg) in a timely manner, which is well-suited for a large number of samples and also when adequate sample collection is a limiting factor. The protocol isolates not only DNA from various plant species but also RNA. This makes it highly effective for molecular analyses compared to previously described CTAB methods optimised for DNA isolation. The appropriate concentration of the components enables high-quality DNA and RNA isolation from plant tissues simultaneously. Additionally, this protocol is compatible with commercially available columns. For DNA and RNA to be qualified for next-generation sequencing platforms, the protocol is supplemented with columns to purify either DNA or RNA from the same tissue to meet high standards for sequencing analyses. This protocol provides an ideal approach to overcome potential obstacles in isolating high-quality DNA or RNA from a wide range of plant species for downstream molecular analysis.
Competing Interests: The authors have declared that no competing interests exist.
(Copyright: © 2023 Masoomi-Aladizgeh et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)
Databáze: MEDLINE
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