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…

INTEGRAPE guidelines and tools: an effort of COST Action CA17111

INTEGRAPE was a European interdisciplinary network for “data integration to maximize the power of omics for grapevine improvement” (CA17111, https://integrape.eu/), funded by the European COST Association from September 2018 to 2022. This Action successfully developed guidelines and tools for data management and promoted the best practices in grapevine omics studies with a holistic future vision of: “Imagine having all data on grapevine accessible in a single place”.

Read More

Study of fungal and bacterial laccases for the reduction of ochratoxin A content in model wine

Ochratoxin A (OTA) is a mycotoxin produced by several filamentous fungi infecting grape bunches (Penicillium and Aspergillus spp.), this toxin pass to must when grapes are crushed and later it is found in wine. Following the evaluations of the toxicity of OTA, European Commission Regulations have been promulgated introducing upper limits for OTA concentrations in various commodities (cereals, cereal products, dried vine fruit, coffee, wine, grape juice, baby foods and dietary foods for special medical purposes).

Read More

Consumo hídrico de la vid, c.v. Listán negro, en la comarca de Tacoronte-Acentejo. Tenerife

Durante el bienio 1998-1999 se estudió el uso consuntivo de cultivos de viña var. Listán negro, en cuatro fincas situadas en la Comarca de Tacoronte-Acentejo, en la isla de Tenerife.

Read More

Identifying physiological and genetic bases of grapevine adaptation to climate change with maintained quality: Genome diversity as a driver for phenotypic plasticity  (‘PlastiVigne’ project)

In the face of climate change, new grapevine varieties will have to show an adaptive phenotypic plasticity to maintain production with erratic water resources, and still ensure the quality of the final product. Their selection requires a better knowledge of the genetic basis of those traits and of the elementary processes involved in their variability. ‘PlastiVigne’, an emblematic project of the Vinid’Occ key challenge, funded by the Occitanie Region (France), tackles this issue with innovative genomic and physiological tools implemented on a unique panel of grape genetic resources representing the genetic diversity of Vitis vinifera. A graph-pangenome is developed from a representative set of high-quality genomes to study the extent and impact of structural genome variations and chromosomal rearrangements in the rapid adaptation capacity of grapevine.

Read More

Applications pratiques du zonage vitivinicole

Le zonage vitivinicole présente toute une série d’applications pratiques. Son importance est en train d’augmenter, soit en fonction des moyens techniques chaque fois plus performants, qui rendent possible le développement des zonages de plus en plus intégrées, consistants et utiles, soit en fonction d’un marché de plus en plus mondialisé. L’article situe la contribution du zonage au niveau de la production vitivinicole et du développement du territoire.

Read More