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…

Metabolomic profiling of heat-stressed grape berries 

The projected rise in mean air temperatures together with the frequency, intensity, and length of heat waves in many wine-growing regions worldwide will deeply impact grape berry development and quality. Several studies have been conducted and a large set of molecular data was produced to better understand the impact of high temperatures on grape berry development and metabolism[1]. According to these data, it is highly likely that the metabolomic dynamics could be strongly modulated by heat stress (HS).

Read More

ANTHOCYANINS EXTRACTION FROM GRAPE POMACE USING EUTECTIC SOLVENTS

Grape pomace is one of the main by-products generated after pressing in winemaking.Emerging methods, such as ultrasound-assisted extraction with eutectic mixtures, have great potential due to their low toxicity, and high biodegradability. Choline chloride (ChCl) was used as a hydrogen bond acceptor and its corresponding hydrogen bond donor (malic acid, citric acid, and glycerol: urea). Components were heated at 80 °C and stirred until a clear liquid was obtained. Distilled water was added (30 % v/v). A solid-liquid ratio of 1 g pomace per 10 ml of eutectic solvent was used.

Read More

Ceramic imprint in wine: influence of hydraulic ratio on ceramic dissolution and wine pH in amphorae systems

This interaction is primarily due to an acidic attack on the ceramic by the wine. It results in (1) the dissolution of the ceramic into the wine and the release of a wide variety of elements; and (2) an increase of the wine pH. The extent of these effects depends on the mineralogical and chemical composition of the ceramic, as well as the hydraulic ratio of the ceramic-wine system (the term hydraulic ratio (ρ) defines here the volume of wine over the surface area of the ceramic in contact with the wine).

Read More

Building of a hierarchy of wines based on terroirs: an initiative from the producers of Muscadet

The Muscadet area is situated in the southeast of Nantes, close to the Atlantic coast. It constitutes the western extension
of the French vineyard “Loire Valley”. The Muscadet is renowned and often spontaneously linked to a white wine.
However it remains misconceived as an ordinary wine, lacking authenticity.

Read More

Kimmeridgian age in Chablis: a geological argument for the social building of a terroir

Situated at the beginning of the 20th century on the territory of the Chablis municipality, delimited according to specialists of the time to plots of “kimmeridgian” origin, the vineyard producing Chablis

Read More