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…

Correlation between agronomic performance and resistance gene in PIWi varieties in the field

Today’s viticulture faces a considerable challenge dealing with fungal diseases and limitations on the use of plant protection products (PPP) have increased the pressure to find more sustainable alternatives. One strategy may be the development and cultivation of disease-resistant grapevine varieties (PIWI) that could maintain crop productivity and quality while reducing dependence on PPP. In this work a set of 9 PIWI varieties (5 white and 4 red) deploying genes for resistance to powdery and downy mildew were evaluated in two consecutive years in Valdegón, La Rioja, with Tempranillo and Viura as controls. The objective was to correlate agronomic performance and disease incidence with the presence of disease resistance genes in two different seasons: with (2023) and without disease pressure (2022).

Analytical and Chemometric Investigation of Phenolic Content of South African Red Wines

Phenolic compounds have been the focus of a lot of research in recent years for their important contribution to sensory characteristics of wine, their beneficial health effects, as well as the possibility they offer of characterising wines. In this contribution, a method is developed that allows the direct injection of wine samples followed by liquid

La zonazione viticola e i compiti dell’amministrazione regionale

Solo attraverso un adeguato intervento di estirpazione e reimpianto dei vigneti è possibile preservare, adeguare e valorizzare il patrimonio viticolo e le produzioni che da esso derivano.
Il reimpianto dei vigneti è pertanto da intendersi come una normale pratica agricola, alla pari della rimonta di stalla in campo zootecnico, ma può assumere toni problematici quando, come si verifica adesso in Toscana per una serie di circostanze legate alla profonda trasfor­mazione della viticoltura avvenuta negli ultimi 30 anni, troppi impianti giungono contem­poraneamente a fine ciclo produttivo e devono essere rinnovati.

Under-row low competitive herbaceous cover: A sustainable alternative to herbicide in vineyards

Weeds are undesirable plants in agroecosystems as they compete with the crop for essential resources such as light, water and nutrients, compromising the final yield and its quality.

Withering of the ‘Moscato giallo’ grapes under covered space

For the purpose of producing predicate wines in northern part of Croatia, grapes are traditionally left on the vine unpicked. However, grapes on the vine are exposed to unfavorable environmental conditions that affect rapid rotting and attacked by birds. To eliminate the mentioned risks, the grapes can be picked and placed in a protected space (loft, greenhouse, etc.) suitable for drying. This study presents the results of research on withering grapes of the ‘Moscato giallo’ variety in two tretment: sun drying (under covered terrace) and drying in the shade (loft). The following quality parameters were monitored: mass of grapes, sugar concentration, content of total acids, pH, content of organic acids.