Big data from small tissues: extraction of high-quality RNA for different plant tissue types during oilseedBrassicaspp. seed development for RNA-Sequencing

Abstract

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 transcriptome during development. 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 gynoecia wall tissue were also collected at the pre-fertilization stage. After testing several RNA extraction methods, E.Z.N.A. Plant RNA Kit and Picopure RNA Isolation kit extraction methods with some modifications, as well as the use of PVPP for seed coats and endosperms at green stages, resulted in high RNA concentrations with clear 28S and 18S bands and high RIN values. Here, we present efficient and reliable RNA extraction methods for different genotypes of Brassica spp for different tissue types during seed development. The high-quality RNA obtained by using these methodologies is suitable for RNA-Sequencing and gene expression analyses.Abbreviations: HSE: Heart stage embryo, HSEnd: heart stage endosperm, HSSC: heart stage seed coat, TSE: torpedo stage embryo, TSSC: torpedo stage seed coat, TSEnd: torpedo stage endosperm, GSE: green stage embryo, GSEnd: green stage endosperm, GSSC: green stage seed coat, MSE: mature stage embryo, MSSC: mature stage seed coat.

Research Article Evaluation of extraction methods for obtaining high-quality RNA from sweet potato

“…The RNA integrity can be evaluated through the use of agarose gels (Azizi et al, 2017;Murgan et al, 2020), as well as through the determination of the RIN (Siles et al, 2020;Vennapusa et al, 2020). However, the integrity analyzed by agarose gels depends on the interpretation of images by the user, which is prone to errors, differently the RIN approach, where an algorithm is used to standardize the RNA quality control, generating an integrity number (Schroeder et al, 2006).…”

“…Several studies have been performed in different plant species, such as citrus (Huma et al, 2020), brassica (Siles et al, 2020), cassava (Behnam et al, 2019), Phaseolus vulgaris (Acosta-Maspons et al, 2019), Elaeis guineensis (Ong et al, 2019), and coffee (Paula et al, 2012;Huded et al, 2018) aiming to establish the best method for obtaining high-quality RNA. Plant tissues may have compounds, such as polysaccharides and polyphenols, that can negatively interfere in isolating high-quality nucleic acids (Badai et al, 2020), turning this process many times unique for each plant tissue.…”