Abstract
BackgroundObtaining 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.ResultsSeeds 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, 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 seed coats and endosperm at green stages 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.ConclusionsHere, 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.
Highlights
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
Seeds are complex structures formed by three tissue types derived from different ontogenetic origins [2] that are subject to changes in the abundance of transcripts from fertilization to seed maturity
Plant RNA kit and modifying the amount of ground tissue, the RB buffer ratios as well as the final elution volume, RNA of highquality and yield was obtained from ovules, ovary walls, Green stage embryo (GSE) or Mature stage embryo (MSE) and Heart stage endosperm (HSEnd) and Torpedo stage endosperm (TSEnd) (Table 2)
Summary
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. To the best of our knowledge, there is only one study in Arabidopsis thaliana that has investigated seed development at a high level of detail, focusing on different tissue types and subregions during seed development [8]. This is the case for Brassica spp, where studies have focused only on the whole seed, specific embryo stages or the late phases of seed development, mainly related to oil biosynthesis and storage seed reserves [9,10,11,12,13,14]
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