terclim by ICS banner
IVES 9 IVES Conference Series 9 Optimized protocol for high-quality RNA extraction from grape tissues using sorbitol pre-wash

Optimized protocol for high-quality RNA extraction from grape tissues using sorbitol pre-wash

Abstract

Obtaining high-quality RNA from grape tissues, including berry pulp, berry skins, stems, rachis, or roots, is challenging due to their composition, which includes polysaccharides, phenolic compounds, sugars, and organic acids that can negatively affect RNA extraction. For instance, polyphenols and other secondary metabolites can bind to RNA, making it difficult to extract a pure sample. Additionally, RNA can co-precipitate with polysaccharides, leading to lower extraction yield. Also, sugars and organic acids can interfere with the pH and ionic properties of the extraction buffer. To address these challenges, we optimized a protocol for RNA isolation from grape tissues. Although commercial kits can provide a rapid extraction, they were inefficient for these plant materials. Similarly, protocols that work well for other vegetal tissues were also inefficient and time-consuming on grape tissues. To overcome these limitations, we added a sorbitol pre-wash step to both a three-day long protocol based on LiCl precipitation and a commercial kit. Our results showed that the addition of a sorbitol pre-wash improved multiple parameters: the A260/280 absorbance ratio, integrity and quality (IQ), and RNA integrity number (RIN). Sorbitol played a crucial role in ensuring high-quality RNA extraction from grape tissues. It inhibits RNase, thereby preserving RNA integrity and stability. It also helps in disrupting cellular membranes, facilitating the release of RNA, and maintains the osmotic pressure through hypertonicity, which is beneficial to RNA extraction. By using sorbitol, commercial kits can be used to extract RNA from challenging grape tissues, leading to an efficient and time-saving procedure.

DOI:

Publication date: June 13, 2024

Issue: Open GPB 2024

Type: Poster

Authors

Annalisa Prencipe1, Antonella Salerno1,2, Marco Vendemia2, Carlo Bergamini2, Margherita D’Amico2, Lucia Rosaria Forleo2, Teodora Basile2, Maria Francesca Cardone2, Antonio Domenico Marsico2, Riccardo Velasco2, Mario Ventura1, Flavia Angela Maria Maggiolini2*

1 Department of Biosciences, Biotechnology and Environment, University of Bari “Aldo Moro”, 70125 Bari, Italy
2 Council for Agricultural Research and Economics – Research Center Viticulture and Enology (CREA-VE), Via Casamassima 148-70010 Turi (Ba), Italy

Contact the author*

Keywords

Vitis vinifera, RNA, sorbitol, extraction protocol

Tags

IVES Conference Series | Open GPB | Open GPB 2024

Citation

Related articles…

Update knowledge about the presence of condensed tannins in grapes and their contributions to astringency perception

Condensed tannin is a principle group of polyphenol compounds derived from grape, greatly contributing to the bioactivity and the sensory perception of wine. Condensed tannins present as a heterogeneous mixture in nature involving various degrees of both polymerization and galloylation. Even though multiple attempts focusing on fractionation of grape condensed tannins by solid-phase have been conducted over the past decades, few individual tannins have been purified and identified. Hence, our knowledge on grape and wine condensed tannin moleculars has to be limited at the several known monomeric, dimeric and trimeric proanthocyanidins

Enzyme treatments during pre-fermentative maceration of white winegrapes: effect on volatile organic compounds and chromatic traits

Volatile organic compounds (VOCs) are very important for the characterisation and quality of the final white wine. An oenological practice to increase the extraction of aroma compounds is the cold pre-fermentative maceration [1,2], although it may also release phenolic compounds that confer darker chromatic traits to white wines, not appreciated by consumers. This practice could be improved by the use of enzymes in order to facilitate the release of the odorous molecules. In this study, the effect of different enzyme treatments during skin contact on the chromatic characteristics and volatile composition of white musts from four winegrape varieties was evaluated.

Exploring the prevalence of esca-induced leaf symptoms in French vineyards and the role of climate: a national scale analysis

Esca, a severe trunk disease affecting vineyards, is caused by fungal pathogens that induce wood necrosis and decay, leaf symptoms, yield losses, and potentially a rapid death of the vine. The prevalence of this disease varies across years, regions, cultivars, and plot ages. Despite its significance in understanding and predicting dieback risk in different vineyards, the role of climate in trunk diseases remains a relatively unexplored research area. While some studies have demonstrated the impact of certain climatic conditions on the prevalence of the disease, they often focus on a limited number of plots and yield conflicting results.We conducted a statistical analysis, using a Bayesian approach on a national database comprising prevalence data of esca from over 500 different plots in France, spanning the years 2003 to 2022 and encompassing various cultivars.

Characterization of bunch compactness and identification of associated genes in a diverse collection of cultivars of Vitis vinifera L.

Compactness is a complex trait of V. vinifera L. and is defined ultimately by the portion of free space within the bunch which is not occupied by the berries. A high degree of compactness results in poor ventilation and consequently a higher susceptibility to fungal diseases, diminishing the quality of the fruit. The easiness to conceptualize the trait and its importance arguably contrasts with the difficulty to measure and quantify it. However, recent technical advancements have allowed to study this attribute more accurately over the last decade. Our main objective was to explore the underlying genetics determining bunch compactness by applying updated phenotyping methods in a collection of V. vinifera L. cultivars with a wide genetic diversity.

Quantifying water use diversity across grapevine rootstock-scion combinations

Vines require proper light levels, temperature, and water availability, and climate change is modifying these factors, hampering yield and quality. Despite the large diversity of rootstocks, varieties, and clones, we still lack knowledge of their combined effects and potential role in a warmer and dryer future. Therefore, we aim to characterize some of the existing diversity of rootstocks and genotypes and their interaction at the eco-physiological level, combining stomatal conductance (gs) and chlorophyll a fluorescence analysis.