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…

Les AOC : un frein ou un moteur à l’innovation ? Retour sur l’histoire économique, scientifique, sociale et technique des vins d’appellation au XXe siècle en France

At a time when the world’s winegrowing industry is having to adapt to a number of challenges, winegrowers are wondering about the consequent changes they will have to make (grape varieties, changes in vineyard and cellar techniques). For winegrowers and consumers alike, there is also the question of how these changes will affect the taste of their wines. This research, based on the study of numerous sources and archives from the 20th century, some of which have never been published before, aims to show that, in the recent past, the winegrowing world has shown incredible resilience in the face of crises, and that the taste and perception of fine wines has changed considerably in 100 years.

Read More

Identifying best parameters to characterize genotypes capability of retaining adequate malic acid at harvest and in final wines

Under current climate change pressures, obtaining grapes with adequate acidity at harvest is one of the main challenges for growers, especially if the goal is producing sparkling wines. This issue arises from two main occurrences: i) higher temperatures enhance degradation of malic acid; ii) grape maturity may occur under suboptimal climatic conditions due to an advanced phenology.

Read More

Influence of the vintage, clone and rootstock on the chemical characteristics of Syrah tropical wines from Brazil

In the Northeast of Brazil, vines can produce twice a year, because annual average temperature is 26ºC, with high solar radiation and water availability for irrigation.

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

Sustaining grape production under challenging climate change circumstances

Grapevines are an important economic crop grown in temperate climates of both hemispheres characterized by short‐term heat spells and heat waves

Read More