Simple and efficient isolation of high-quality total RNA from Hibiscus tiliaceus, a mangrove associate and its relatives.

Hibiscus tiliaceus, one of the mangrove associates, is an ideal plant for the study of ecological adaptation and salt tolerance as it may give clues to the evolutionary pathway by which the highly specialized adaptive syndrome of mangrove has been achieved. This species has extremely high contents of polysaccharides, secondary metabolites and especially polyphenolics in its various tissues, such as leaves and roots. So it is extremely difficult for RNA extraction from its various tissues, which prevents following molecular operations and expression-level studies like microarray and RT-PCR. Traditional methods based on CTAB can produce high-quality RNA from specific species or tissue, but they don't work well in H. tiliaceus. We describe here a modified CTAB-based method using PVP and PVPP with sequential extraction of chlorophorm and acid phenol and then selective salt precipitation, which can constantly isolate high-quality RNA from various tissues of H. tiliaceus within short time. RNA quality was assessed through reverse transcription and RT-PCR experiments, indicating that it could be suitable for a number of downstream purposes. We have already constructed a full-length cDNA library with total RNA isolated through the modified method and successfully used it in the following microarray experiments, which is aimed to unravel ecological adaptation and salt tolerance of H. tiliaceus. Moreover, we showed that this method could also be successfully applied to other plants in Malvaceae sensu lato (including Sida, Pachira, Sterculia, Urena, and Abelmoschus) with very abundant polyphenols and polysaccharides.