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.

Download the abstract

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…

Identification of important genomic regions controlling resistance to biotic and abiotic stresses in Vitis sp. through QTL meta-analysis

In the context of global change, the environmental conditions are expected to be more stressful for viticulture. The choice of the rootstock may play a crucial role to improve the adaptation of viticulture to new biotic and abiotic threats (Ollat et al., 2016). However, the selection of interesting traits in rootstock breeding programs is complex because of the combination of multiple targets in a same ideotype. In this sense, the integration of studies about the genetic architecture for desired biotic and abiotic response traits allow us to identify genomic regions to combine and those with interesting pleiotropic effects.

Read More

French regulations related to vineyard spraying and examples of devices developed in France and around the world to limit the risks of point-source pollution

Managing pests in vineyards presents a major challenge for winegrowers, who are seeking effective solutions to control diseases and pests.

Read More

Fungal communites diversity and functional roles of different types of Botrytis cinerea infected grape berries on different growing sites

Botrytis cinerea, an Ascomycota pathogen with a broad host range, infects over 1200 plant species. Grapes infected by this pathogen, which subsequently develop a noble rot, remain in the vineyard for an extended period, thus being exposed to a diverse array of physical, chemical and biological factors, which give rise to a complex microbial community.

Read More

Determination of steviol glycosides in wine by HPLC

The SCL laboratory in Bordeaux is one of the two official control laboratories for wine and wine products in france, under the authority of the ministry of finance and two of its general directorates: the DGCCRF (directorate general for competition, consumer affairs and fraud control) and the DGDDI (directorate general of customs and excise duties). In this capacity, it verifies the regulatory compliance of wines and investigates any possible falsifications or fraud. Steviol glycosides are natural sweeteners that are not authorized as additives in wine.

Read More

La zonazione della D.O.C. Bolgheri (Castagneto C.): aspetti metodologici ed applicativi

The results of the first step of the zoning study carried out in Bolghery appellation area (Castagneto Carducci, Tuscany) in the 1993-1995 period have been recently published. Quality factors of Bolgheri appellation and different “terroirs ” were identified.

Read More