Jazowska Justyna, Przyborowski Mateusz, Wojciechowski Marek, Groszyk Jolanta
Plant Breeding and Acclimatization Institute-National Research Institute, Radzików, 05-870 Błonie, Poland.
Int J Mol Sci. 2024 Dec 8;25(23):13188. doi: 10.3390/ijms252313188.
RNA quality and integrity are critical for many studies in plant molecular biology. However, extracting high-quality RNA from cereal grains is challenging due to the presence of polysaccharides, polyphenols, and other compounds that bind or coprecipitate with RNA particles. To address this, we introduced an initial purification step into the Tri Reagent Solution protocol, which effectively eliminated starch and other contaminants. The performance of this modified protocol was then compared with five other RNA extraction methods, including those based on Tri Reagent Solution and the Direct-zol RNA Miniprep Plus Kit. Introducing our method modification prior to homogenization with Tri Reagent Solution successfully yielded total RNA with both high quality (RIN values ranging from 9.50 to 9.70) and high efficiency, making it suitable for both mRNA extraction using the Dynabeads mRNA Purification Kit and library preparation for transcriptome sequencing by long-read methods, such as Oxford Nanopore Technologies. The protocol was successfully applied to total RNA extraction from rye grains at 14 and 21 days after pollination. This study demonstrates that improving the Tri Reagent Solution protocol through initial purification enables the extraction of high-quality RNA from rye grains.
RNA的质量和完整性对于植物分子生物学的许多研究至关重要。然而，由于存在多糖、多酚和其他与RNA颗粒结合或共沉淀的化合物，从谷物中提取高质量RNA具有挑战性。为了解决这个问题，我们在Tri Reagent Solution方案中引入了一个初始纯化步骤，该步骤有效地去除了淀粉和其他污染物。然后将这个改进方案的性能与其他五种RNA提取方法进行比较，包括基于Tri Reagent Solution的方法和Direct-zol RNA Miniprep Plus试剂盒。在使用Tri Reagent Solution匀浆之前引入我们的方法改进，成功获得了高质量（RIN值在9.50至9.70之间）和高效率的总RNA，使其适用于使用Dynabeads mRNA纯化试剂盒提取mRNA以及通过长读长方法（如牛津纳米孔技术）进行转录组测序的文库制备。该方案已成功应用于授粉后14天和21天的黑麦籽粒总RNA提取。这项研究表明，通过初始纯化改进Tri Reagent Solution方案能够从黑麦籽粒中提取高质量RNA。
