Isolation of high quality RNA and construction of a suppression subtractive hybridization library from ramie (Boehmeria nivea L. Gaud.)

Abstract

Isolation of high quality RNA from ramie (Boehmeria nivea L. Gaud.) is difficult due to its high levels of polyphenols, polysaccharides, pectin, fat, wax and other secondary metabolites. A modified procedure based on guanidinium isothiocyanate for RNA preparation of ramie was developed in this study. High concentrations (5%, v/v) of guanidinium isothiocyanate, PVP-4000, sodium citrate and sodium lauryl sarcosinate and beta-mercaptoethanol were used in the extraction buffer, together with a low pH sodium acetate (pH 4.0) added to improve the RNA quality. The average yield was about 400 microg RNAg(-1) fresh leaves. One SSH library which was induced by ramie anthracnose was constructed by utilizing the RNA extracted through the present method. These results showed that our protocol was applicable for RNA isolation from recalcitrant ramie tissues.