Abstract:
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 optimized 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 et al., (2004). The changes included an increased rate or use of Extraction Buffer (EB) for every
gram ground leaf tissue (20ml EB per 1 gram tissue), incubation of the Tissue-EB and Chloroform: Isoamyl
alcohol (24:1) mixture at a lower water-bath temperature of 50oC 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 μg), high A260:A280 and A260:A230 ratios (> 2.0) and high
integrity (distinct and visible 28S and 18S rRNA bands) from young and old cassava leaves, compared to
RNA (from the same leaf tissues) generated by several other published methods.
