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…

Viticultura protegida: uso de mallas sombreadoras fotoselectivas como una herramienta para enfrentar la crisis climática en uva de mesa en el norte de Chile

The production of table grapes in Chile is of great importance, being one of the main established fruit crops with over 43,000 hectares distributed across a diverse climate range, from the southern limit of the Atacama desert to the mediterranean zone. Chile is also one of the leading exporters of table grapes. producers must confront the challenges posed by the climate crisis, such as decreased rainfall, increased heatwaves, and extreme temperature events during the growing season, mainly associated with desertification in northern Chile (Atacama and Coquimbo regions).

Updating the Winkler index: An analysis of Cabernet sauvignon in Napa Valley’s varied and changing climate

This study aims to create an updated, agile viticultural climate index (similar to the Winkler Index) by performing in-depth analyses of current and historical data from industry partners in several major winegrowing regions. The Winkler Index was developed in the early twentieth century based on analysis of various grape-growing regions in California. The index uses heat accumulation (i.e. Growing Degree Days) throughout the growing season to determine which grape varieties are best suited to each region. As viticultural regions are increasingly subject to the complexity and uncertainty of a changing climate, a more rigorous, agile model is needed to aid grape growers in determining which cultivars to plant where. For the first phase of this study, 21 industry partners throughout Napa Valley shared historical phenology, harvest, viticultural practice, and weather data related to their Cabernet sauvignon vineyard blocks. To complement this data, berry samples were collected throughout the 2021 growing season from 50 vineyard blocks located throughout 16 American Viticultural Areas that were then analyzed for basic berry chemistry and phenolics. These blocks have been mapped using a Geographic Information System (GIS), enabling analysis of altitude, vineyard row orientation, slope, and remotely sensed climate data. Sampling sites were also chosen based on their proximity to a weather station. By analyzing historical data from industry partners and data specifically collected for this study, it is possible to identify key parameters for further analysis. Initial results indicate extreme variability at a high spatial resolution not currently accounted for in modern viticultural climate indices and suggest that viticultural practices play a major role. Using the structure of data collection and analyses developed for the first phase, this project will soon be expanded to other wine regions globally, while continuing data collection in Napa Valley.

A fast and sensitive method for total tannin determination in wine based on the substoichiometric quenching of silicon-rhodamine conjugates

Tannins are chemically diverse polyphenols contributing to important sensory attributes of food and beverages. In wine, their structure and quantity depend on several factors, such as the grape variety, climate, soil, viticultural and enological practices and the wine-aging process.

Hyperspectral imaging for the appraisal of varietal aroma composition along maturation in intact Vitis vinifera L. Tempranillo Blanco berries

The knowledge of the grape aromatic composition during ripening provides very important information for winegrowers, who may carry out different viticultural practices, or determine the harvest date more accurately. However, there are currently no tools that allow this measurement to be carried out in a non-invasive and rapid way. For this reason, the aim of this work was to design a non-invasive methodology, based on hyperspectral imaging to estimate the aromatic composition and total soluble solids (TSS) of Tempranillo Blanco berries during ripening.

Novel table grape varieties as “ready-to-eat” products

Consumers are increasingly requesting ready-to-eat products, which are time-saving and convenient. Offering ready-to-eat fruits and vegetables represents a quick and easy way for any consumer to add healthy products to their diet. In this study, we evaluated the aptitude of several table grape varieties to be included in the processing and packaging lines of ready-to-eat products. The following work was based on the characterization of genetic materials and varietal innovation.