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…

Effect of SO2, GSH and gallotannins on the shelf-life of a cortese white wine

Studying the effect of the addition of reduced glutathione (GSH) and/or gallotannins at bottling to limit the use of SO2 in white winemaking.

Aromas of Riesling wine: impact of bottling and storage conditions

Storage temperature and bottling parameters are among the most important factors, which influence the development of wine after bottling. It is well studied that higher storage temperatures speed up chemical reactions and results in faster wine aging [1,2]. It is also known that higher SO2 level and lower oxygen content provide better protection and longer shelf-life for the wine. At the same time, the mechanisms of chemical transformations of wine aromas during the aging process are not fully understood. In particular, how oxidation reactions contribute to the transformations of varietal aroma compounds.In the present study [3], we investigated the development of Riesling wine depending on a series of bottling conditions, which differed in the free SO2 level in wine (low—13 mg/L, medium—24 mg/L, high—36 mg/L), CO2 treatment of the headspace.

Overview on wine and health 32 years after the French paradox 

Phenolic compounds or polyphenols are the most abundant and ubiquitous secondary metabolites present in the plant kingdom with more than 8000 phenolic structures currently known. These compounds play an important role in plant growth and reproduction, providing protection against biotic and abiotic stress such as pathogen and insect attack, UV radiation and wounding. (poly)phenols are widely distributed in the human diet mainly in plant-derived food and beverages (fruits, vegetables, nuts, seeds, herbs, spices, tea and red wine).

Impact of polyclonal selection for abiotic stress tolerance on the yield and must quality traits of grapevine varieties

The effects of climate change in viticulture are currently a major concern, with heat waves and drought affecting yield, wine quality, and in extreme cases, even plant survival. Ancient grapevine varieties have high intravarietal genetic variability that so far has been explored successfully to improve yield and must quality. Currently, there is little information available on intravarietal variability regarding responses to stress. In the current work, the intravarietal genetic variability of several Portuguese varieties was studied for yield, must quality, and tolerance to abiotic stress, through indirect, rapid, and nondestructive measurements carried out in the field.

Toasting and grain effect on Tempranillo red wine aged in Quercus petraea barrels

The barrel-making process is widely recognized as a crucial practice that affects the composition of barrel-aged wine. After the drying process, the staves are considered ready for barrel assembly, which includes the processes of bending and toasting the barrel structure. Toasting is considered one of the most critical stages in determining the physical and chemical composition of the staves, which can influence the chemical and sensory composition of the wine aged in barrels made from them [1].