Extraction of high-quality tissue-specific RNA from Londonplane trees (Platanus acerifolia), permitting the construction of a female inflorescence cDNA library

Abstract

The London plane tree (Platanus acerifolia Willd.) has global importance as an urban landscaping tree and is the subject of genetic-improvement programs for productive sterility, disease and/or insect resistance. Molecular analysis techniques are crucial to such programs, but may be impeded by specific difficulties encountered during nucleic acid isolation. A detailed RNA isolation and purification protocol, based on established cetyltrimethyl-ammonium bromide (CTAB) extraction techniques combined with additional purification steps using butanol and the ionic detergent CTAB, which overcomes these problems in the woody species P. acerifolia, was conducted. In short, phenolic compounds are bound to soluble polyvinylpyrrolidone and then separated out through LiCl precipitation of the RNA. Subsequently, protein- and carbohydrate-contaminants are removed by chloroform partitioning followed by LiCl-mediated precipitation. The resulting isolates of RNA were found to be of sufficient quality for successful use in reverse transcription PCR analysis. Furthermore, RNA isolates from female inflorescences were used for the construction of a cDNA library. This library was found to contain several full-length cDNA clones of MADS-box genes, consistent with the library being representative of inflorescence expression profiles.