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…

Studio dell’ambiente viticolo attraverso la parametrazione (punto di incrocio) delle curve di maturazione delle uve (pinot nero, oltrepo’ pavese pv italia settentrionale – 45° parallelo Nord)

Sono stati presi in considerazione alcuni dati agrometeorologici dell’Oltrepò Pavese (temperature e piovosità degli ultimi 80 anni) e gli studi delle curve di maturazione condotti in zona sul Pinot nero da spumante negli anni (1988-1991, 1999-2000, 2006-2008), si nota che l’aumento progressivo negli anni delle temperature attive (indice di Winkler) ha determinato un anticipo dell’invaiatura, definita dal parametro “punto di incrocio” (intersezione delle funzioni di zuccheri ed acidità nel tempo), con conseguente anticipo della data di vendemmia di circa 12-15 gg.

Evaluating alternatives to cold stabilization in wineries: the use of carboximethyl cellulose, potassium polyaspartate, electrodialysis and ion exchange resins – the results after one year in the bottle

The tartaric stabilization of wines before bottling to avoid the precipitation of tartaric acid salts is an important and common step during wine production. The presence of precipitated salt crystals in bottle wines is detrimental for their quality and even a legal issue in some countries. Cold stabilization is the most common stabilization treatment. Although it has been shown to be effective, it has some significant disadvantages, mainly regarding losses of color and aromas and its high cost. Therefore, other products and methodologies are being introduced in the wineries for the replacement of this process. Some of these new techniques involve the reduction of the ions causing the insolubilization of tartaric acid while other are based in the formation of protective colloids or the inhibition of the crystallization of salts. In this study, white, rosé and red wines have been treated with carboxymethylcellulose, potassium polyaspartate and an ion exchange resin. The tartaric stability of the wines, together with the oenological, chromatic and sensory characteristics were studied after the wines had been stored during one year in the bottle. The results indicate that the use of carboxymethyl cellulose and potassium polyaspartate maintained the best the sensory and chromatic characteristics and the wine stability of the wines in comparison with an untreated control wine.

Aroma characterization of aged cognac spirits: contribution of volatile terpenoid compounds

Cognac spirit aromas result from the presence of a wide variety of volatile odorous compounds associated with the modalities of distilled spirit elaboration and during aging. Indeed, these odorous compounds play an essential role in the finesse and complexity of the aged Cognac.

Tracking of sulfonated flavanol formation in a model wine during storage

The aim of this work was to determine the reaction products of bisulfite with grape seed flavanols and changes therein over different storage conditions in a model wine

High-power ultrasound for improving chromatic characteristics in wines. Does a varietal effect exist?

The use of high-power ultrasound (US) during the winemaking process has been extensively studied at laboratory scale in order to demonstrate its possible use to improve the extraction of compounds of interest. However, studies on semi-industrial and industrial scale are needed to confirm this positive effect, since the International Organization of Vine and Wine approved its industrial use in 2019 [1].