Isolation of functional RNA from grapevine tissues poor in nucleic acid content

A modified procedure for efficient isolation of intact and biologically active RNA from leaves, berries and cell cultures of grapevine (Vitis vinifera L.) is described. RNA is extracted in the presence of mild denaturants followed by sequential precipitations with potassium acetate and isopropanol. RNA is recovered free of contaminants after centrifugation through a CsCl cushion. Alternatively, RNA from grapevine callus and berry cell suspensions can be purified after the isopropanol step by high-salt precipitations, without a CsCl centrifugation step. The resulting RNA is of high-quality, as judged by agarose gel electrophoresis and northern blot analysis. Furthermore, RNA isolated by this method is biologically active as was evidenced by in vitro translation and analysis of the products by polyacrylamide gel electrophoresis and fluorography. This procedure can also be applied to other perennial woody plant species.