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…

Analysis of the interaction of melatonin with glycolytic proteins in Saccharomyces cerevisiae during alcoholic fermentation 

Melatonin is a bioactive compound with antioxidant properties, that has been found in many fermented beverages, such as beer and wine [1]. Indeed, it has been shown that yeast can synthesize melatonin during alcoholic fermentation, although its role inside the cell, as well as the metabolic pathway involved in its synthesis, is still unclear [1]. Recent studies showed that during fermentation, melatonin interacts with different proteins of the glycolytic pathway in both Saccharomyces and non-Saccharomyces yeast, for instance glyceraldehyde 3-phosphate dehydrogenase, pyruvate kinase or enolase [2].

The influence of RNAi-expressing rootstocks in controlling grey mold on grapevine cultivars

Worldwide, with an average of 6.7 million cultivated hectares, of which exclusively 51% in Europe (faostat, 2021), the production of table and wine grapes is a leading sector, with continued growth in Europe in the area devoted to vine cultivation. during the growing season, most of the plant organs can be susceptible to several fungal and oomycete diseases, leading to important economic losses and causing detrimental effects on fruit quality. the increasingly scarce availability of fungicidal products, often also related to their relative impact on the environment, coupled with the emergence of resistance in the pathogen to these products, make defence increasingly challenging.

Effects of winemaking variables on the chemical and sensory quality of Schiava wines up to one year storage in bottle

The interactive effects of three major enological variables were evaluated on the quality of Schiava wine up to one year of storage in bottle.

The limonene-derived mint aroma compounds in red wines. Recent advances on analytical, chemical aspects and sensory aspects

In recent years, the ageing bouquet of red Bordeaux wines has been partially unveiled by a chemical and sensory point of view1–3. Minty and fresh notes were found to play a key role in the definition of this complex concept, moreover the freshness dimension in fine aged red wines plays an important role in typicity judgement by wine professionals

Early detection project – make a GTD infection visible without disease symptoms

The presence of grapevine trunk diseases (GTDs) related pathogens leads to severe economic losses in wine‐growing regions all over the world