An Optimised, Small-scale Preparation of High-quality RNA from Dry Seeds of Davidia involucrata

Introduction - The dormancy of Davidia involucrata seeds normally lasts for an extended period of time and because of this unique property the species is an excellent model for studying the molecular mechanisms of plant dormancy. The use of minimal plant material is desirable for RNA extraction since D. involucrata is a rare plant and it is relatively difficult to collect large amounts of seeds in order to perform molecular biology studies. Objective - To improve the quality of RNA obtained from seeds of D. involucrata by eliminating the oxidation of polyphenols during extraction and by preventing polysaccharides and other impurities from being extracted. Methods - A previously described method was modified by the addition of 4% (w/v) poly(N-vinyl-2-pyrrolidone) to the dry seeds during grinding and by adding 5% (v/v) beta-mercaptoethanol and 28% (v/v) ethanol to the extraction buffer. Two further centrifugation steps (5000 and 8000 rpm) were utilised and one-seventh volume of ethanol was incubated with the supernatant at 4 C for 2-3 h prior to the precipitation of RNA. Results - Following these modifications, an effective method was established for total RNA extraction from a small amount of dry seeds of D. involucrata. The isolated RNA was shown to have high purity and integrity by gel electrophoresis and spectrophotometry, and was confirmed to be suitable for RT-PCR and the construction of cDNA libraries. Conclusion - The modified method reduced the amount of seeds required for extraction of total DNA and was beneficial for preserving the endangered species. Copyright (C) 2008 John Wiley & Sons, Ltd.