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…

Genome editing applications on grapevine cv. Aglianico for the knockout of susceptibility genes related to fungal diseases

Context and purpose of the study. Italy hosts diverse grapevine varieties crucial for viticultural biodiversity. Preserving this biodiversity is essential for maintaining a diversified genetic pool and addressing future challenges such as climate change and emerging plant diseases.

MAPPING THE CONCENTRATIONS OF GASEOUS ETHANOL IN THE HEADSPACE OF CHAMPAGNE GLASSES THROUGH INFRARED LASER ABSORPTION SPECTROSCOPY

Under standard wine tasting conditions, volatile organic compounds (VOCs) responsible for the wine’s bouquet progressively invade the glass headspace above the wine surface. Most of wines being complex water/ethanol mixtures (with typically 10-15 % ethanol by volume), gaseous ethanol is therefore undoubtedly the most abundant VOC in the glass headspace [1]. Yet, gaseous ethanol is known to have a multimodal influence on wine’s perception [2]. Of particular importance to flavor perception is the effect of ethanol on the release of aroma compounds into the headspace of the beverage [1].

Studio per la caratterizzazione delle produzioni vitivinicole dell’area del Barbera d’Asti DOC

Il Barbera rappresenta sicuramente uno dei più importanti vitigni autoctoni del Piemonte occu­pando circa il 50% della superficie vitata regionale. Esso è ancora diffuso su un’area molto vasta, che si estende per oltre 200.000 ha, dando origine a diverse produzioni vinicole tutelate da denominazioni d’origine.

Study to optimize the effectiveness of copper treatments for low impact viticulture

Among all pathologies that afflict grapevine, Downy Mildew (DM) is the most important. Generally controlled using Copper (Cu), recently European Commission confirmed its usage but limiting the maximum amount to 28 Kg per hectare in 7 years (Reg. EU 2018/1981).

Effect of rootstock and preplant fumigation on plant parasitic nematode development in Washington wine grapes

In Washington State, the majority of winegrape (Vitis vinifera) vineyards are planted to their own roots. This practice is possible due to the lack of established phylloxera populations, and is preferred due to the ease of retraining after damaging winter cold events. However, own-rooted V. vinifera is generally susceptible to most plant parasitic nematodes that attack grape. In Washington State, management of nematodes is dominated by preplant soil fumigation. One practice that may mitigate economic loss due to nematodes is the adoption of nematode-“resistant” rootstocks.