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…

Delayed irrigation nearly doubles yield loss compared to anthocyanin gain in Southern Oregon Pinot noir

Irrigation initiation timing is a critical annual management decision that has cascading effects on grapevine productivity and wine quality.

Read More

Assessing bunch architecture for grapevine yield forecasting by image analysis

It is fundamental for wineries to know the potential yield of their vineyards as soon as possible for future planning of winery logistics. As such, non-invasive image-based methods are being investigated for early yield prediction. Many of these techniques have limitations that make it difficult to implement for practical use commercially. The aim of this study was to assess whether yield can be estimated using images taken in-field with a smartphone at different phenological stages. The accuracy of the method for predicting bunch weight at different phenological stages was assessed for seven different varieties.

Read More

IMPACT ON CHITOSAN APPLICATION OF DIFFERENT MICROORGANISMS HAVING OENOLOGICAL INTEREST

Chitosan is an effective antimicrobial agent available in the wine industry, because it ensures the control of a of spoilage microorganisms, such as Brettanomyces of lactic acid bacteria.

Read More

Using open source software in viticultural research

Many high quality Open Source scientific applications have been available for a long time. Some of them have proved to be particularly useful for carrying out the usual activities involved in viticultural research projects, such as statistical analyses (including spatial analyses), GIS work, database management (possibly integrated with statistical and spatial analysis) and even “low-level” often highly time-consuming activities (e.g. repetitive task on text files).

Read More

Free amino acid composition of must from 7 Vitis vinifera L. cv. in Latium (Italy)

Free amino acid concentrations in must of 7 Vitis vinifera cultivars (Cabernet Franc, Syrah, Merlot, Montepulciano, Sangiovese, Cesanese d’Affile, Carmenere) grown in the Latium region (Italy) were monitored from 2003 to 2005. The cultivars were located in a homogeneous soil and climatic zone and with the same training system (Cordon Spur).

Read More