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…

The influence of vine row position in terraced Merlot vineyards on water deficit and polyphenols – case study in the Vipava Valley, Slovenia

A study was conducted in the Vipava Valley (Slovenia) to understand the effects of positioning rows of Merlot (Vitis vinifera L.) vines on terraces on plant available water, yield, and grape composition

Read More

Soils, climate, nutritive status and production of cv “Palomino fino” in the superior quality area of the Jerez-Xérès-Sherry zone

The Registered Appellation of Origin Mark (RAOM) « Jerez-Xérès-Sherry and Manzanilla Sanlucar de Barrameda » is one of the oldest and more important zone in wine history and production. «Albarizas» unit (white calcareous marls with sea-fossils) is the most representative geological material of the RAOM (75%) and even more in the central-NW area of the RAOM, known as «Jerez Superior» area (Superior Quality Sherry Area). « Albarizas » form undulated hillocks (3-10% slope) and hills (>10% slope), the litologic unit has E-W and S-W direction, and Regosols and Leptosols are the principal soils.

Read More

Monitoring the tawny port wine aging process using precision enology

AIM: Tawny Port wine is produced in the Douro Demarcated Region by blending several fortified wines in different aging stages. During the aging process in small wood barrels, the red wine color progressively develops into tawny, medium tawny, or light tawny.

Read More

Influence of spraying of copper fungicides on physiological parameters of Vitis vinifera L. Cv. ‘Merlot’

Vine downy mildew is one of the most frequent diseases in intensive vineyards. Bordeaux mixture (B.m.), in order to control the disease has been applied onto vineyards since the end of the 19th century. The intensive use of Cu-fungicides could influence the physiology of grapevine. It is also possible that high amounts of foliar Cu sprays trigger stress responses in vine leaves.

Read More

Utilizing ozone for the management of powdery mildew (Erysiphe necator Schwein.) in vineyards: potential and challenges

Powdery mildew, caused by fungal pathogens, poses a significant threat to grapevines in the DOCa Rioja region. In efforts to improve control strategies while reducing reliance on conventional phytosanitary products, ozone could constitute a potential alternative. However, it has short persistence, thus requiring frequent treatments. This study aimed to assess the suitability of ozone as an active substance for controlling powdery mildew within a phytosanitary strategy aimed at reducing conventional phytosanitary product usage. The strategy integrating ozone with conventional products yielded powdery mildew levels comparable to conventional treatments in both disease incidence and severity.

Read More