Isolation of RNA of high quality and yield from Ginkgo biloba leaves

Abstract

An improved protocol was developed to isolate total RNA in good yield and integrity from Ginkgo biloba leaves containing high levels of flavonoid glycosides, terpene lactones, carbohydrates and polyphenolic secondary metabolites. Polyvinylpolypyrrolidone at 2% and β-mercaptoethanol at 4% were added to the standard CTAB extraction buffer and, after chloroform and phenol extraction, the pellet obtained by ethanol/acetate precipitation was washed and a second phenol/chloroform extraction was introduced to remove co-precipitated polysaccharides. Both A₂₆₀/A₂₃₀ and A₂₆₀/A₂₈₀ absorbancy ratios of isolated RNA were around 2 and the yield was about 0.4 mg g^--1 fresh weight. At least seven distinct rRNA bands were detected by denaturing gel electrophoresis. Sharp hybridization signals were obtained from Northern blots with both nuclear and plastid gene probes. Two gene fragments: nuclear-encoded cab and chloroplast encoded rbcL were successfully amplified by RT-PCR, suggesting the integrity of isolated RNA. The total RNA isolated by this protocol is of sufficient quality for subsequent molecular applications.