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…

Ultra-High Pressure Homogenization (UHPH): a technique that allows the reduction of SO2 in winemaking

Ultra-High Pressure Homogenization (UHPH) is an innovative, efficient and non-thermal technology that can be applied at different stages in winemaking in order to reduce or avoid the use of sulphites. During 2022 vintage, a batch of Xarel·lo must was processed by UHPH at 300 MPa with an inlet temperature (Ti) of 4 ºC. In order to verify the influence of the UHPH treatment in wine characteristics, alcoholic fermentations with this must (UHPH) were carried out and compared with a control batch (without SO2 addition (C)) and a sulphited batch, in which 60 mg/L of total SO2 (SO2) were added.

The sensory features of the landscapes

When someone watches a hilly landscape, the image beauty creates emotions and frames of mind not easily forgettable, but sometimes man’s intervention by means of soil movement and reduction of the natural biodiversity can significantly modify the landscape and consequently the above-mentioned emotions. One speculates if sensory appreciation of a wine may be strongly affected by psychological factor: landscape beauty.

Agronomic behavior of three grape varieties in different planting density and irrigation treatments

In the O Ribeiro Denomination of Origin, there is a winemaking tradition of growing vines under a high-density plantation framework (8,920 vines/ha) and maintaining its vegetative cycle under rainfed conditions.
Currently, viticulture is advancing to plantation frames in which the density is considered medium (5,555 vines/ha), thus allowing mechanized work to be carried out for vineyard management operations. Although, the application of irrigation applied proportionally to the needs of the vegetative cycle of the vine, is a factor that increasingly helps a good development of the vine compared to the summer period, with increasingly uncertain weather forecasts.

Parcours de découverte des terroirs viticoles

A partir des recherches conduites sur la caractérisation des terroirs viticoles par des chercheurs de l’Unité de Recherches Vigne et Vin (1, 2, 3, 4, 5) du Centre INRA d’Angers, Terre des Sciences, le Centre de Culture Scientifique et Technique d’Angers (CCSTA) a mis au point un parcours de découverte d’une journée dans le vignoble angevin avec une approche pluridisciplinaire.

Prise en compte de la notion de terroir dans les AOC en France : Aspects Culturels

“The vine and the wine are great mysteries. Only the vine makes us intelligible what is the true flavor of the earth”. Colette. The notion of terroir has always been the basis of the notion of AOC from which it is inseparable. It is moreover the definition of the production zone which was at the start of the attempts to set up the designation of origin, at the beginning of the century, after the phylloxera crisis.