Precise isolation of high-quality RNA from leaves and storage roots of cassava (Manihot esculenta) for gene expression studies.

Abstract

Abstract The isolation of RNA from leaves and storage roots of cassava (Manihot esculenta) is a challenging one, due to the presence of large amounts of polyphenolic compounds, polysaccharides, and tuber proteins. RNA with high quality and intact integrity is vital for gene expression studies. We hereby report a precise, reproducible, and less cumbersome technique for isolating high-quality RNA from leaves and storage roots of cassava with minimal contamination from polyphenols, polysaccharides, and other secondary metabolites, using affordable reagents. This protocol functions without guanidinium salts in the extraction buffer. The presence of guanidinium salts usually leads to the formation of agglomerates during the extraction of RNA from plant tissues with high starch contents. RNA was isolated from leaves and storage roots of 10 different casava genotypes which yielded between 1576.1 to 2861.9 µg/ml for RNA isolated from the leaf tissues and 2761.2 to 3873.5 µg/ml for RNA isolated from the storage roots. The A260:A280 ratios of the total RNA were more than 2.0 for both leaf and storage root samples, indicating minimal contamination from polysaccharides and polyphenols. The RNA samples recorded intact integrity, as demonstrated by clear 28 S and 18 S rRNA bands observed on agarose gel electrophoresis. The RIN values ranged between 7.2 to 8.0. Also, the RNA samples were successfully used for transcriptome sequencing. The present method which yielded high-quality and transcriptionally competent RNA samples is suitable for use in gene expression studies and downstream applications in molecular breeding of cassava and related root/tuber crops.