An Optimised Cetyltrimethylammonium Bromide (CTAB)-Based Protocol for Extracting RNA from Young and Old Cassava Leaves

Abstract

Ribonucleic acid (RNA) integrity, quality and quantity are critical in most plant molecular studies. Extracting high quality RNA from cassava leaves and other recalcitrant plant tissues are difficult due to the presence of polysaccharides, polyphenols and other secondary metabolites that often co-precipitate with the final RNA extract. This is an optimised a CTAB-based method that suitably extracts RNA from the polysaccharide-rich cassava leaves. The modifications were introduced into a version of the CTAB protocol as described by Gasic and colleagues [1]. The changes included an increased rate or use of Extraction Buffer (EB) for every gram ground leaf tissue (20 ml EB per 1 gram tissue), incubation of the Tissue-EB and Chloroform: Isoamyl alcohol (24:1) mixture at a lower water-bath temperature of 50°C and all centrifugation steps carried out at 4°C. In addition, the EB contained a higher concentration of soluble polyvinylpyrrolidone (PVP-K-30). The pH of sodium acetate was lowered to 5.2 and a final two-step high molarity (10M) Lithium Chloride (LiCl) precipitation was applied. Ethyl alcohol concentration was raised to 100%. The modified CTAB method produced RNA of high concentration (>1.0 μg), high A260:A280 and A260:A230 ratios (> 2.0) and high integrity (distinct and visible 28S rRNA and 18S rRNA bands) from young and old cassava leaves, compared to RNA (from the same leaf tissues) generated by several other published methods or commercial kits. The protocol is efficient, simple, and reproducible and is therefore recommended for RNA extraction from metabolite-rich cassava leaves or plants with similar tissues.