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…

Aroma compounds involved in the fruity notes of red wines potentially adapted to climate change.

Currently, climate change represents one of the major issues for the wine sector. The increasing temperature already recorded and expected in the upcoming years reduce the vegetative cycle of the grape varieties planted in Bordeaux area, affecting the physicochemical parameters of grapes and consequently, the quality of wine. From a sensory point of view, the attenuation of the fresh fruity character in some varietals is accompanied by the accentuation of dried-fruit notes [1]. As a new adaptive and ecological strategy on global warming, some winegrowers have initiated changes in the Bordeaux blend of vine varieties using late-ripening grape varieties [2]. 

Phenotypic variations of primary metabolites yield during alcoholic fermentation in the Saccharomyces cerevisiae species

Saccharomyces cerevisiae, as the workhorse of alcoholic fermentation, is a major actor of winemaking. In this context, this yeast species uses alcoholic fermentation to convert sugars from the grape must into ethanol and CO2 with an outstanding efficiency: it reaches on average 92% of the maximum theoretical yield of conversion. Moreover, S. cerevisiae is also known for its great genetic diversity and plasticity that is directly related to its living environment, natural or technological and therefore to domestication. This leads to a great phenotypic diversity of metabolites production.

Design of microbial consortia to improve the production of aromatic amino acid derived compounds during wine fermentation

Wine contains secondary metabolites derived from aromatic amino acids (AADC), which can determine quality, stability and bioactivity. Several yeast species, as well as some lactic acid bacteria (LAB), can contribute in the production of these aromatic compounds. Winemaking should be studied as a series of microbial interactions, that work as an interconnected network, and can determine the metabolic and analytical profiles of wine. The aim of this work was to select microorganisms (yeast and LAB) based on their potential to produce AADC compounds, such as tyrosol and hydroxytyrosol, and design a microbial consortium that could increase the production of these AADC compounds in wines.

Effects of post-fermentative cold maceration on chemical and sensory characteristics of Syrah, Cabernet Franc and Montepulciano wines

Astringency sensation decreases slowly during the aging of red wine. Complex reactions of condensation and precipitation of wine polyphenols are involved in this phenomenon. Wine composition and conditions of aging, such as temperature and oxygen availability, strongly influence evolution of the phenol matrix. Recently, a Post-Fermentative cold Maceration (PFM) technique was tested with the aim of accelerating reactions leading to the reduction of astringency and exploiting chemical compounds not extracted from the solid parts of grapes during the previous traditional maceration phase. To this purpose, an innovative maceration system was engineered and used to perform PFM trials on marc derived from vinification of different varieties of red grapes.

Red wine substituted esters involved in fruity aromatic expression: an enantiomeric approach to understand their sensory impact and their pathway formation

Among red wines ethyl esters, those from short hydroxylated and branched-chain aliphatic acids constitute a family with a particular behavior and sensory importance. They have been previously discussed in the literature [1] and recent studies have established that some of them were strongly involved in of red wines’ fruity aroma [2]. As some among them have an asymmetrical carbon atom, it seemed important to separate their different enantiomers to obtain an accurate assessment of their organoleptic impact. Three chiral esters have been identified, presenting alkyl and/or hydroxyle substituants: ethyl 2-hydroxy-4-methylpentanoate, ethyl 2-methylbutanoate, and ethyl 3-hydroxybutanoate.