Šķipars, V., Šņepste, I., Krivmane, B., Veinberga, I. and Ruņģis, D. 2014. A Method for Isolation of High-quality Total RNA from Small Amounts of Woody Tissue of Scots Pine. Baltic Forestry 20(2): 230-237

Isolation of large amounts of high quality RNA from a limited amount of starting material is particularly difficult if extracting from woody tissues of trees. Often the amount of starting material may be limited, when specific tissues are harvested. There are no commercially available kits specifically developed and intended for isolation of RNA from wood. RNA isolation from wood is difficult in general, and the high amount of resins and other secondary metabolites in wood of Scots pine (Pinus sylvestris L.) make the isolation of nucleic acids particularly difficult. Due to the large amounts of resins and other viscous substances in woody tissues of Scots pine, the use of column based extraction techniques is risky because the columns can clog leading to total loss of irreplaceable sample and, in general, the concentration of extracted RNA is low. In our experience, solution-based commercial kits like TRI Reagent (Life Technologies) and cetyltrimethylammonium bromide (CTAB)-based extraction protocols for isolation of RNA from small amounts of wood also did not yield sufficient amounts of RNA for further analysis. Our objective was to develop an improved method for RNA extraction from small amounts of woody tissue of Scots pine (Pinus sylvestris L.). We describe a protocol for isolation of RNA using the Thermo Scientific Genomic DNA isolation kit with a modified protocol, which has been used to consistently isolate high quality RNA from small amounts of mature Scots pine phloem and xylem in our laboratory. The extracted RNA is of sufficient quantity and quality for most down-stream applications, including gene expression studies and high-throughput transcriptome sequencing. In addition, the extracted RNA also contained sufficient concentrations of micro RNA and other small RNAs for further analysis.

Key words: RNA isolation, woody tissue, Scots pine, Pinus sylvestris L.