Big data from small tissue extraction of high-quality RNA for RNA-Sequencing from different oilseed Brassica seed tissues during seed development

A - Papers appearing in refereed journals

Siles-Suarez, L., Eastmond, P. J. and Kurup, S. 2020. Big data from small tissue extraction of high-quality RNA for RNA-Sequencing from different oilseed Brassica seed tissues during seed development . Plant Methods. 16, p. 80.

AuthorsSiles-Suarez, L., Eastmond, P. J. and Kurup, S.
Abstract

Background
Obtaining high-quality RNA for gene expression analyses from different seed tissues is challenging due to the presence of various contaminants, such as polyphenols, polysaccharides and lipids which interfere with RNA extraction methods. At present, the available protocols for extracting RNA from seeds require high amounts of tissue and are mainly focused on extracting RNA from whole seeds. However, extracting RNA at the tissue level enables more detailed studies regarding tissue specific transcriptomes during seed development.
Results
Seeds from heart stage embryo to mature developmental stages of Brassica napus and B. oleracea were sampled for isolation of the embryo, endosperm and seed coat tissues. Ovules and ovary wall tissue were also collected from pre-fertilized buds. Subsequent to testing several RNA extraction methods, we determined that modifications applied to E.Z.N.A. Plant RNA and Picopure RNA Isolation kit extraction methods resulted in RNA with high yield and quality. Furthermore, the use of polyvinylpolypyrrolidone for the extraction of RNA from seed coats and endosperm at green seed stage resulted in high-quality RNA. As a result of the introduced modifications to established RNA extraction methods, the RNA from all the above-mentioned tissues presented clear 28S and 18S bands and high RIN values, ranging from 7.0 to 10.0. The protocols reported in this study are not only suitable for different and challenging seed tissue types, but also enable the extraction of high-quality RNA using only 2 to 3 mg of starting tissue.
Conclusions
Here, we present efficient, reproducible and reliable high-quality RNA extraction methods for diverse oilseed Brassica spp reproductive tissue types including pre-fertilization and developing seed tissues for diploid and polyploid species. The high-quality RNA obtained is suitable for RNA-Sequencing and subsequent gene expression analysis.

KeywordsRNA extraction; Plant; Difficult tissue; Seed; Embryo; Endosperm; Reproductive; Seed coat; Brassica
Year of Publication2020
JournalPlant Methods
Journal citation16, p. 80
Digital Object Identifier (DOI)doi:10.1186/s13007-020-00626-0
Open accessPublished as bronze (free) open access
FunderBiotechnology and Biological Sciences Research Council
Funder project or codeBrassica Rapeseed And Vegetable Optimisation (BRAVO)
Tailoring Plant Metabolism ISP
Publisher's version
Accepted author manuscript
Output statusPublished
Publication dates
Online05 Jun 2020
Publication process dates
Accepted28 May 2020
PublisherBiomed Central Ltd
ISSN1746-4811

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