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…

Influence of two yeast strains and different nitrogen nutrition on the aromatic compounds in Lugana wine

Lugana Protected Designation of Origin (PDO) wines are made from Turbiana grapes. The aroma of Lugana wines results from the combined contribution of esters, terpenes, norisprenoids, sulfur compounds and the benzenoid methyl salicylate. This study aims to investigate how volatile aroma compounds are affected by different nitrogen supplies and yeast strains. Wines were produced with a standard protocol with 2021 Turbiana grapes with two different yeasts Zymaflore Delta e Zymaflore X5 (Laffort, France).During the alcoholic fermentation of the must, when H2S appeared, additions of various nitrogen supply were made: inorganic nitrogen, organic nitrogen, a mix of inorganic and organic nitrogen and organic nitrogen with an addition of pure methionine. During wine fermentation, a daily measurement of hydrogen sulfide was carried out.

Productivity, quality, and thermal needs of the Piedirosso vine: four years of observations

The effects of temperature on cv Piedirosso, indigenous of the Campania region (South of Italy), were tested in order to study its possible influence on grapevine and to discover how to optimize the qualitative expression

Phenolic characterization of four different red varieties with “Caíño” denomination cultivated in Northwestern Spain

In this work, these four red varieties were characterized in terms of phenolic composition. Thus, the anthocyanin accumulation and the extractability evolution during ripening were compared.

Occurrence of methyl salicylate in lugana wines: aroma impact and biogenesis 

AIM Methyl salicylate (MeSA) has been reported as a potentially impactful compound in Verdicchio wines produced in central Italy. Lugana is another white wine produced in the north-east of Italy from a grape locally known as Trebbiano di Soave, sharing a very similar genetic background with Verdicchio. The aims of this study were evaluating MeSA occurrence in Lugana, assessing its aroma impact on white wines aroma and elucidating its biogenesis during vinification. METHODS Fifteen Lugana wines were analysed for methyl salycilate content in comparison with Verdicchio, Pinot grigio and Garganega wines. MeSA impact on white wine aroma was studied by means of triangular test, adding MeSA at different concentrations. Possible routes of MeSA formation by yeast were investigated by means of a high throughput assay in which S. cerevisiae cells were put in contact with precursor such as salicylic acid (esterification) or glycosidic extracts (glycosidase). Sub-fractions of Lugana glycosidic extracts were also obtained by HPLC fractionation, allowing further evaluation of precursors role.

Effect of moderate wine consumption in animal models

In 1979, the so-called “french paradox” was proposed, that is, a correlation between wine consumption, a diet rich in saturated fats, and a low mortality from coronary heart disease. On the other hand, it has also been described that alcohol consumption has negative effects on aging and increases the risk of liver cirrhosis and cancer. However, both hypotheses are based on population studies that may present distortions due to multiple factors (geographic, diet, smoking, socioeconomic level, etc.).