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…

Non-destructive his based analysis for shelf-life evaluation of table grape 

Fast, accurate, and non-destructive analytical techniques based on hyperspectral imaging (hsi) represent effective tools for food quality evaluation. A visible change in the appearance of a fresh product often negatively impacts the perceived quality from a consumer’s point of view.

Read More

An overview of the impact of clone, environmental factors and viticultural techniques on rotundone concentration in red wines

Rotundone is the main aroma compound responsible for peppery notes in red wine. This positive and very potent molecule has an odor threshold of 8 ng/L in water and 16 ng/L in red wine. It has been detected in several grape varieties with some of the highest concentrations recorded in Syrah, Duras, Tardif and Noiret, an interspecific hybrid grown in the North-East of the USA. If several winemaking practices have been identified to lower rotundone in wine, up to date, no enological solution has proved its efficiency to maximize it. This means that efforts to produce high rotundone wines must be undertaken in vineyards. This work provides practical ways that can be used by winegrowers to modulate rotundone levels in their wines.

Read More

Cell wall remodeling mediated by specific PME genes plays a role in grapevine response to Botrytis cinerea

Botrytis cinerea (Bc) is one of the main pathogens affecting the cultivated grapevine. A key role in grapevine tissue colonization is played by cell wall (CW) remodeling driven by CW Modifying Enzymes (CWMEs), expressed both by the host and the pathogen. Their action can impact CW integrity and trigger specific immune signaling, thus influencing Bc infection outcome. To further characterize the role of the CW in the grapevine response to Bc, two contrasting genotypes in their resistance to the fungus were artificially inoculated at full bloom. RNA-seq analysis and biochemical characterization of the CW and its modification in samples collected at 24 hours post-inoculation highlighted significant differences between genotypes.

Read More

Pesticide – Free viticulture: towards agroecological wine-producing socio-ecosystems

Can we cultivate grapevine without pesticides? This is a huge challenge for this emblematic crop, which is one of the largest users of plant protection products. Pesticides are mainly used to protect the vine against leaf diseases (powdery mildew, mildew, black-rot), even in organic farming, which uses copper in particular. What are the research avenues that can help eliminate pesticides today?

Read More

Vino e paesaggio: materiali per il governo del territorio vitivinicolo. Il piano regolatore delle città del vino

S’intende per Piano Regolatore delle Città del Vino la metodologia per redigere la parte strutturale dello strumento comunale di governo del territorio. Parliamo, infatti, del principale strumento comunale di governo del territorio, così come è venuto maturando nella riflessione delle Città del Vino, strumento che si misura con la sfida di governare tutto il territorio in modo coerente e sostenibile, a partire dal riconoscimento del valore del “sistema vigneto” e della sua intrinseca fragilità.

Read More