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…

Tools for terroir classification for the grape variety Kékfrankos

A 3-year study was carried out in order to evaluate the ecophysiology, yield and quality characteristics of Vitis vinifera L. cv. Kékfrankos (syn. Limberger) at Eger-Nagyeged hill (steep slope) and at Eger-Kőlyuktető (flat) vineyard sites located in the Eger wine region, Hungary.

Read More

Early fermentation aroma profiles of grape must produced by various non-Saccharomyces starters

Saccharomyces cerevisiae is the most commonly used yeast species in winemaking. The recent research showed that non-Saccharomyces yeasts as fermentation starters show numerous beneficial features and can be utilized to reduce wine alcoholic strength, regulate acidity, serve as bioprotectants, and finally improve wine aromatic complexity. The majority of published studies on this topic investigated the influence of sequential or co-inoculations of non-Saccharomyces and S. cerevisiae yeasts on the aroma of final wine.

Read More

Using open source software in viticultural research

Many high quality Open Source scientific applications have been available for a long time. Some of them have proved to be particularly useful for carrying out the usual activities involved in viticultural research projects, such as statistical analyses (including spatial analyses), GIS work, database management (possibly integrated with statistical and spatial analysis) and even “low-level” often highly time-consuming activities (e.g. repetitive task on text files).

Read More

HYBRID GRAPEVINE CV BACO BLANC, BETWEEN TRADITION AND MODERNISM: FOCUS ON ENDOGENOUS EUGENOL AS RESISTANCE FACTOR TO BOTRYTIS CINEREA

The well-known antifungal and antibiotic molecule, eugenol, is widely spread in various plants including clove, basil and bay. It is also abundant in the hybrid grapevine cultivar (cv) Baco blanc (Vitis vi-nifera x Vitis riparia x Vitis labrusca), created by François Baco (19th century) in the Armagnac region. This study confirmed this cv as highly resistant to Botrytis cinerea by comparing fruit rot incidence and severity with two Vitis vinifera cultivars: Folle Blanche and Ugni Blanc. We have demonstrated the efficiency of eugenol in vitro, by further investigating the effect of small concentrations of eugenol, 3 to 4 ppm (corresponding to IC10), on B. cinerea. By comparing the two major modes of action (direct or volatile antibiosis), the vapour inhibiting effect of eugenol was more powerful. In the skin of Baco blanc berry, the total eugenol concentration reached a maximum at veraison, i.e. 1118 to 1478 μg/kg.

Read More

Sélection génétique des variétés originelles d’Arménie, berceau de la viticulture mondiale

Armenia, a small country in the South of the Caucasus, has been rediscovering its wine-growing past since the discovery in 2007 of archaeological wine-growing remains dating back around 8,000 years. They are among the oldest in the world. Despite a great diversity of grape varieties, Armenian winegrowers did not have sufficiently organized genetic collections to produce plants and satisfy the growing demand for planting.

Read More