An Improved RNA Extraction Protocol for Rye Grain Full-Length Transcriptome Sequencing

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.