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. 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, 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. 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.

中文翻译：

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来自小组织的大数据：在种子发育过程中从不同油籽芸苔种子组织中提取用于 RNA 测序的高质量 RNA。

由于存在各种污染物，例如干扰 RNA 提取方法的多酚、多糖和脂质，因此从不同的种子组织中获取用于基因表达分析的高质量 RNA 具有挑战性。目前，从种子中提取 RNA 的可用方案需要大量组织，主要集中在从整个种子中提取 RNA。然而，在组织水平提取 RNA 可以更详细地研究种子发育过程中的组织特异性转录组。对欧洲油菜和甘蓝从心脏阶段胚胎到成熟发育阶段的种子进行取样，以分离胚胎、胚乳和种皮组织。还从受精前的芽中收集胚珠和卵巢壁组织。在测试了几种 RNA 提取方法之后，对大肠杆菌进行了修改。ZNA 植物 RNA 和 Picopure RNA 分离试剂盒提取方法产生了高产量和高质量的 RNA。此外，在绿色阶段将聚乙烯聚吡咯烷酮用于种皮和胚乳会产生高质量的 RNA。由于对已建立的 RNA 提取方法进行了修改，来自上述所有组织的 RNA 呈现出清晰的 28S 和 18S 条带和高 RIN 值，范围从 7.0 到 10.0。本研究报告的方案不仅适用于不同且具有挑战性的种子组织类型，而且仅使用 2 至 3 mg 的起始组织即可提取高质量的 RNA。在这里，我们提出了高效，可重复且可靠的高质量 RNA 提取方法，适用于多种油籽芸苔属繁殖组织类型，包括预受精和二倍体和多倍体物种的种子组织发育。获得的高质量 RNA 适用于 RNA 测序和后续基因表达分析。