Versatile and efficient RNA extraction protocol for grapevine berry tissue, suited for next generation RNA sequencing

Abstract

Background and Aims: New high-throughput tools for transcriptomic analysis such as RNA sequencing have been developed rapidly in recent years. These technologies provide new opportunities for biologists to improve the understanding of gene expression underlying important physiological processes. The sequencing of RNA by nextgeneration technologies is, in particular, dependent on the use of pure and un-degraded RNA. Many fruits from perennial crops share an elevated concentration of tannins and polysaccharides and a strong dilution of cytosolic content upon vacuole hypertrophy. The grapevine berry presents these characteristics together with high vacuole acidity, which has never been taken into account in previous protocols, and poses thereby a particular challenge for RNA extraction. Methods and Results: In order to counterbalance these limitations, we developed a new extraction protocol based on a tri-sodium-citrate extraction buffer that has a high buffer capacity and proved to be particularly suitable for low pH plant tissue. Conclusions: The proposed RNA extraction procedure produces consistently high-quality RNA (RNA integrity number > 8 up to 10; 260 nm/280 nm > 2; 260 nm/230 nm > 1.9) with an elevated yield [20 μg/g fresh mass (FM) for ripe berries up to 150 μg/FM for green berries]. The method was developed initially for berry tissues but proved also to be efficient for other grapevine tissues, such as nodes, roots, leaves, seeds, lignified shoots and flowers. Significance of the Study: The method is most suitable for modern gene expression analysis methods, such as RNA sequencing and microarray studies. Successful construction of cDNA libraries and high numbers of detected reads obtained by next-generation RNA sequencing underline the applicability of the protocol.