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…

Exploring the effect of ripening rates on the composition of aroma and phenolic compounds in Cabernet-Sauvignon wines

The study of cultural practices to delay ripening and the characterization of their effect on wine composition is important in the mitigation of accelerated ripening caused by higher temperatures

Evaluation of uhph treatment as an alternative to heat treatment prior to the use of proteolytic enzymes on must to achieve protein stability in wine

There are currently enzyme preparations on the market with specific protease activities capable of degrading unstable must proteins and preventing turbidity in white and rosé wines. The main drawback is the need to heat the must at 75ºc for 1-2 minutes to denature the proteins and facilitate enzyme action.

Enhancing sustainability in viticulture through digital technologies: A case study from Smyrnakis winery

The integration of digital technologies in vineyard management offers substantial opportunities for enhancing sustainability, efficiency, and grape quality.

Autochthonous non-Saccharomyces extra-cellular metabolism of tryptophan, tyrosine, and phenylalanine

Amino acids are crucial nitrogen sources in yeast metabolism, influencing both biomass production and fermentation rate. The breakdown byproducts of amino acids contribute to the aroma of the wine and wine’s health benefit compounds. This study focused on the yeast’s extracellular metabolic profile of tryptophan, tyrosine, and phenylalanine belonging to the group of aromatic amino acids in experimental Maraština wines. Alcoholic fermentations were conducted on sterile grape Maraština must using seven autochthonous non-Saccharomyces yeasts in sequential fermentation with commercial Saccharomyces cerevisiae.

Winter physiology in a warmer world: Cold hardiness and deacclimation sensitivity drive variation in spring phenology

As the climate warms, the focus of concern in viticulture often turns to how higher temperatures may shift growing regions, change the character of AVAs, and alter fruit quality. However, climate warming is increasing most quickly during the winter dormancy cycle, a critical and often underappreciated portion of the grapevine life cycle. In response to decreasing temperatures and decreasing daylength, grapes initiate a series of physiological changes to enter dormancy, acquire freeze resistance, and time spring phenology such that the growing season begins after threat of frost.