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 Yeast Derivative Products on Aroma compounds retention in model wine

For many years, enological research has developed commercial formulates of yeast derivatives as stabilizing agents and technological adjuvants in winemaking. These products are obtained from yeast by autolytic, plasmolytic, or hydrolytic processes that liberate many macromolecules from the yeast cell, principally polysaccharides and oligosaccharides and most specifically mannoproteins that are well known for their ability to improve tartaric stability and to reduce the occurrence of protein hazes (Ángeles Pozo-Bayón et al., 2009; Charpentier & Feuillat, 1992; Morata et al., 2018; Palomero et al., 2009).

Towards a spatial analysis of antique viticultural areas: the case study of Amos (Turkey) and some other places

Interpretation of ancient texts, such as the Amos epigraphic farming leases, questions both locations and spatial extents of the viticultural area, as well as soils, landscapes, cropping methods

Effect of oenological tannins on wine aroma before and after oxidation: a real-time study by coupling sensory (TDS) and chemical (PTR-ToF-MS) analyses

Polyphenols are important compounds involved in many chemical and sensory wine features. In winemaking, adding oenological tannins claims to have positive impacts on wine stability, protection from oxidation and aroma persistence. Polyphenols are antioxidant compounds by either scavenging reactive oxygen and nitrogen species or chelating Fe2+ ions (1). However, as tannins oxidation leads to the formation of highly reactive species (i.e. ortho-quinones), it is still unclear if they have an effective role toward oxidation of wine aromas (2). In this work, we aim at studying the effect of two commercial tannins (proanthocyanidins, ellagitannins) on red wine flavour (mainly aroma) before and after air exposition.

Long-term flooding effects on the physiological and productive performance of Montepulciano and Sangiovese cultivars

Extreme climatic events, such as prolonged drought followed by intense flooding, increasingly impact viticulture, affecting vine physiology, productivity, and grape composition.

Volatilome in grapevine leaves is defined by the variety and modulated by mycorrhizal symbiosis

Volatile organic compounds (VOCs) constitute a diverse group of secondary metabolites key for the communication of plants with other organisms and for their adaptation to environmental and biotic stresses. The emission of these compounds through leaves is also affected by the interaction of plants with symbiotic microorganisms, arbuscular mycorrhizal fungi (AMF) among them [1]. Our objective was to know the concentration and profile of VOCs emitted by the leaves of two grapevine varieties (Tempranillo, T, and Cabernet Sauvignon, CS, grafted onto R110 rootstocks), inoculated or not with a consortium of five AMF (Rhizophagus irregularis, Funneliformis mosseae, Septoglomus deserticola, Claroideoglomus claroideum and C. etunicatum).