Isolation of functional total RNA from Tilia cordata leaves and pollen

Abstract

Conditions for isolation of high quality total RNA from European linden (Tilia cordata) leaves and pollen were determined. Pure total RNA was isolated from linden leaves utilizing Qiagen plant mini kit while total RNA isolated from linden pollen using this method was degraded. Successful isolation of total RNA from both linden pollen and leaves, however, was achieved following TRIZOL? preparation of total RNA. Total RNA isolated using TRIZOL? was contaminated with genomic DNA but treatment with enzyme DNase, in solution or on-column, efficiently removed the genomic DNA. Furthermore, conditions for elimination of genomic DNA contamination on-column and isolation of pure total RNA from leaves were optimized. Isolated total RNA from both leaves and pollen was used successfully in firstand second-strand cDNA synthesis reactions, as well as, in RT-PCR, demonstrating that the total RNA isolated using this method is functional. In conclusion, pure and functional total RNA from Tilia cordata leaves and pollen (27.8 ? 7.9?g/g leaves; 25.7 ? 1.1?g/g pollen) can be obtained and applicable for further molecular biology studies.