An optimized RNA extraction method for diverse leaves of Hawaiian Metrosideros, a hypervariable tree species complex.

Abstract

The isolation of RNA from trees is challenging due to the interference of polyphenols and polysaccharides with downstream processes. Furthermore, many RNA extraction protocols are time consuming and involve hazardous chemicals. To address these issues, we aimed to develop a safe protocol for high-quality RNA extraction from diverse Metrosideros taxa representing a broad range of leaf toughness, pubescence, and secondary metabolites. We tested popular RNA isolation kits and protocols that were effective on other recalcitrant trees, including a broad range of optimization and purification steps. We optimized a protocol involving two silica-membrane column-based kits that yielded high-quantity RNA with an RNA integrity number >7 and without DNA contamination. All RNA samples were used successfully in a follow-on RNA-Seq experiment. We present an optimized high-throughput RNA extraction protocol that yielded high-quality and high-quantity RNA from three contrasting leaf phenotypes within a hyperdiverse woody species complex.