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…

Studying PIWIs in three dimensions: agronomic, economic and ecological evaluation of 14 fungus-tolerant cultivars in Luxembourg

Growing fungus-tolerant cultivars (PIWIs) reduces the need of fungicide use by 50-80 %. PIWIs have the potential to address climate change adaptation and mitigation simultaneously.

Genetic traceability of the varietal origin of wines: a robust application for must and wines during alcoholic fermentation

Industry and regulatory agencies have developed regulations to ensure authenticity and compliance with wine composition limits. However, this can be truncated by the absence of simple and robust analytical methodologies, uninfluenced by the environment, different oenological techniques and cultural practices. Genetic fingerprinting is the most powerful tool for unequivocal varietal identification; it is not affected by the environment or agronomic practices; however, its usefulness in musts and wines has been controversial and there is currently no routine certification of varietal origin based on DNA analysis.

Yeast interactions in chardonnay wine fermentation: impact of different yeast species using ultra high resolution mass spectrometry

During alcoholic fermentation, when yeasts grow simultaneously, they often do not coexist passively and in most cases interact with each others

Green Vineyards: skills development for wine industry personnel: responding to the challenges of climate change

A fair and sustainable society, with a modern, resource-efficient and competitive economy cannot be achieved without a workforce to support it.

Juvenile-to-adult vegetative phase transition in grapevine 

The sequential activity of miR156 and miR172 controls the juvenile to adult phase transition in many plant species, where miR156 abundance decreases while miR172 increases along plant development. Very little is known about phase transition in horticultural woody species, which show substantially long vegetative phases. In grapevine, phase transition seems to be dissociated, displaying a first transition from juvenile to adult vegetative state in the first year, coincident with tendril differentiation and a subsequent induction of inflorescences in place of some of tendrils in later years under flowering inductive environmental conditions. Since grapevine is a highly heterozygous species, the generation of genetically homogeneous material for replicated transcriptomic analyses from seed-derived plants was a main challenge.