An effective method for extracting high-quality RNA from grapevine

Abstract

Grapevine (Vitis vinifera L.) is one of the most important economic crops in the world. Because of this importance, one finds widespread molecular genetic research on this species, an important element of which is high quality RNA. However, in woody plants, isolation of this molecule is often rather difficult, as the tissues of the various organs may contain significant amounts of secondary metabolites (polysaccharides and polyphenols). These compounds can be removed only with great difficulty during extraction. Our aim was to describe an accurate and usable method for RNA isolation from old leaf tissue starting with a small sample volume (up to 50 mg) to make RNA extraction possible even when younger, developing leaves and shoots are not available. Unlike most related studies, we have also tried to provide a highly detailed methodology to avoid lengthy fine-tuning procedures and thus, to save time and money. The advantages of isolation from leaves include the fast and easy collection of samples and the possibility to study both molecular-genetic changes in physiological processes and plant-pathogen interactions. Our version of the so called CTAB method proved to be efficient in extracting grapevine RNA, which is indicated by the A260:A280 and A260:A230 ratios measured between 2.05 and 2.06 and between 2.06 and 2.09 using an UV-VIS spectrophotometer (NanoDrop 2000, Thermo Fischer Scientific, Massachusetts, USA). The integrity analysis of the RNA was carried out using Agilent RNA 6000 Nano LabChip® (Agilent 2100 Bioanalyzer Califronia, USA). The RIN value (RNA Integrity Number) of the samples was between 7.7 and 8.1, which indicated that small degree of RNA degradation occurred during extraction. The efficiency of the RNA isolation was determined by transcriptional analyses of six housekeeping genes (CYSP, YSL8, Actin, SAND, EF1-α, GAPDH) using quantitative real‐time PCR (qrt-PCR), which was carried out in three biological and two technical replicates applying SYBER Green technology (Qiagen Ltd, Germany). The presence of a single peak in the melt curve and amplification curve analyses and low standard errors between the Ct values indicated the specificity of the primers to bind to the cDNA, with no interference of PCR inhibitors.

Acknowledgments

This research was supported by the National Research, Development and Innovation Office (TKP2021-NKTA-16).