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.

Download the abstract

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…

Vineyard Landscape in Vale dos Vinhedos: Pressure and Protection

The region with Origin Indication Vale dos Vinhedos (IPVV) is located in the cities of Bento Gonçalves, Garibaldi and Monte Belo do Sul. It was established in November 2002, through an act of Instituto Nacional de Propriedade Industrial, according to Resolução INPI Nº. 75, of 2000, and Law Nº. 9.279 of 1996. The changes on the grape growing and wine making in the last 15 years, with the expansion of grapevines Vitis viníferas cultivation, the installation of several modern family wine companies, making wine with competitive quality, among other aspects, have enabled the implementation of concepts of Geographical Indications.

Read More

Sensory and chemical effects of postharvest grape cooling on wine quality

Wine cellars are affected by seasonally fluctuating workloads and face challenges especially in the harvest period connected to the required timely processing of the harvested grapes.

Read More

Understanding novel germplasm solutions: sensory, chemical and preliminary hedonic insights of wines made from Australian first-generation mildew resistant cultivars

One of the major issues for wine production in Australia is the management and eradication of powdery and downy mildews and the associated yield losses they present, costing Australian grape growers upwards of AUD$160M per annum [1].

Read More

EMERGENCE OF INORGANIC PHOSPHONATE RESIDUES IN GRAPEVINE PLANT PARTS, BERRIES AND WINES FROM SOURCES OTHER THAN FOLIAR SPRAYING

Inorganic phosphonates are known to effectively support the control of grapevine downy mildew in vi- ticulture. Their application helps the plant to induce an earlier and more effective pathogen defense. However, inorganic phosphonates have been banned in organic viticulture due to their classification as plant protection products since October 2013. Despite the ban, phosphonate has been recently detected in organic wines.

Read More

Monitoring grapevine water status using Landsat 8 images: a two-year case study in a Merlot vineyard

Viticulture needs for spatial and temporal information are increasing to improve vineyard management, especially concerning water efficiency. Remote sensing, particularly from satellites, can be a powerful tool to assess vineyard characteristics such as vigor or water status in space-time. In this study, we use Landsat 8, an American Earth observation satellite with six bands from the visible (VIS) to the Short-Wave Infrared (SWIR) domains with 30m spatial resolution and two thermal bands with 100m spatial resolution.

Read More