Department of Agronomy, 2004 Throckmorton Plant Sciences Center, Kansas State University, 1712 Claflin Road, Manhattan, KS, 66506-5501, USA.
Department of Molecular and Structural Biochemistry, North Carolina State University, Raleigh, NC, 27695, USA.
Sci Rep. 2020 Oct 9;10(1):16887. doi: 10.1038/s41598-020-73958-5.
Using existing protocols, RNA extracted from seeds rich in starch often results in poor quality RNA, making it inappropriate for downstream applications. Though some methods are proposed for extracting RNA from plant tissue rich in starch and other polysaccharides, they invariably yield less and poor quality RNA. In order to obtain high yield and quality RNA from seeds and other plant tissues including roots a modified SDS-LiCl method was compared with existing methods, including TRIZOL kit (Invitrogen), Plant RNeasy mini kit (Qiagen), Furtado (2014) method, and CTAB-LiCl method. Modifications in the extraction buffer and solutions used for RNA precipitation resulted in a robust method for extracting RNA in seeds and roots, where extracting quality RNA is challenging. The modified SDS-LiCl method revealed intense RNA bands through gel electrophoresis and a nanodrop spectrophotometer detected ratios of ≥ 2 and 1.8 for A260/A230 and A260/A280, respectively. The absence of starch co-precipitation during RNA extraction resulted in enhanced yield and quality of RNA with RIN values of 7-9, quantified using a bioanalyzer. The high-quality RNA obtained was demonstrated to be suitable for downstream applications, such as cDNA synthesis, gene amplification, and RT-qPCR. The method was also effective in extracting RNA from seeds of other cereals including field-grown sorghum and corn. The modified SDS-LiCl method is a robust and highly reproducible RNA extraction method for plant tissues rich in starch and other secondary metabolites. The modified SDS-LiCl method successfully extracted high yield and quality RNA from mature, developing, and germinated seeds, leaves, and roots exposed to different abiotic stresses.
使用现有的方案，从富含淀粉的种子中提取的 RNA 通常会导致 RNA 质量较差，不适合下游应用。虽然有一些方法被提出来从富含淀粉和其他多糖的植物组织中提取 RNA，但它们总是产量较低，RNA 质量较差。为了从种子和其他植物组织（包括根）中获得高产和高质量的 RNA，我们将改良的 SDS-LiCl 方法与现有的方法（如 TRIZOL 试剂盒（Invitrogen）、Plant RNeasy mini 试剂盒（Qiagen）、Furtado（2014）方法和 CTAB-LiCl 方法）进行了比较。改良的提取缓冲液和用于 RNA 沉淀的溶液的改进使得该方法能够在提取质量 RNA 具有挑战性的种子和根中提取 RNA，该方法具有较强的稳健性。改良的 SDS-LiCl 方法通过凝胶电泳显示出强烈的 RNA 条带，纳米分光光度计检测到 A260/A230 和 A260/A280 的比值分别为≥2 和 1.8。在 RNA 提取过程中淀粉不共沉淀，导致 RNA 产量和质量提高，使用生物分析仪检测 RNA 的 RIN 值为 7-9。通过下游应用（如 cDNA 合成、基因扩增和 RT-qPCR）证明了所获得的高质量 RNA 是合适的。该方法还可有效地从包括田间种植的高粱和玉米在内的其他谷物的种子中提取 RNA。改良的 SDS-LiCl 方法是一种从富含淀粉和其他次生代谢产物的植物组织中提取 RNA 的稳健且高度可重复的方法。改良的 SDS-LiCl 方法成功地从成熟、发育和萌发的种子、叶片和根中提取了高产和高质量的 RNA，这些种子、叶片和根暴露在不同的非生物胁迫下。
