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

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 introduc...

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Bibliographic Details
Published in:	PloS one Vol. 18; no. 12; p. e0295852
Main Authors:	Masoomi-Aladizgeh, Farhad, Jabbari, Leila, Khayam Nekouei, Reza, Aalami, Ali, Atwell, Brian J, Haynes, Paul A
Format:	Journal Article
Language:	English
Published:	United States Public Library of Science 14-12-2023 Public Library of Science (PLoS)
Subjects:	Acetic acid Acids Analysis Biology and life sciences Bromides Buffers Cetyltrimethylammonium bromide Chemical tests and reagents Chlorine compounds Deoxyribonucleic acid DNA DNA sequencing Edetic acid Ethylenediaminetetraacetic acid Ethylenediaminetetraacetic acids Evaluation Flowers & plants Gene sequencing Genetic aspects Genetic research Genomes Genomics Homogenization Introduced species Lab Protocol Lysis Metabolites Next-generation sequencing Nucleic acids Nucleotide sequencing Paternity Plant species Plant tissues Plants Protocol Quality control Reagents Research and analysis methods Ribonucleic acid RNA Sodium Sodium chloride Transcriptomics Whole genome sequencing Iran
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Summary:	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.
Bibliography:	ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23 Competing Interests: The authors have declared that no competing interests exist.
ISSN:	1932-6203 1932-6203
DOI:	10.1371/journal.pone.0295852