High-yield RNA extraction from Nepenthes × ventrata pitchers by simple modification of the CTAB method

RNA extraction from some plants is difficult owing to the presence of pigments and secondary metabolites in vacuoles and cell walls, which hinder molecular biological studies. In this study, we developed a simple method for high-yield RNA extraction from pitchers of the carnivorous plant Nepenthes × ventrata that is recalcitrant to RNA isolation. RNA was extracted from pitchers of Nepenthes × ventrata at different developmental stages using conventional methods. Additionally, a modified cetyltrimethylammonium bromide (CTAB) method, in which an ethanol-precipitation step at high LiCl concentration was introduced, was used to extract RNA. Conventional purification methods yielded low or negligible amounts of RNA from pitcher tissues of Nepenthes, and the extracts contained visible pigment contamination. The conventional CTAB method, which is effective for RNA extraction from sweet potato tissues, also yielded only a small amount of RNA from Nepenthes tissues; most of the RNA remained in the supernatant discarded during the LiCl precipitation step, along with the contaminating pigments. By replacing this step with ethanol precipitation in the presence of high concentration of LiCl, the contaminating pigments were removed, and RNA with high purity and yield was recovered. Purified RNA could be efficiently used for full-length cDNA synthesis. The modified method requires no expensive extraction kits and is relatively safe because it does not use phenol. Therefore, this method may be effectively applied to plants that are recalcitrant to RNA isolation.