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…

La sémantique liée à la notion de terroir : une objectivité pluridisciplinaire

It is not easy at first sight to give an exhaustive definition of the notion of terroir as it can be simplified or complicated at will. Thus the vagueness that surrounds this concept leaves the door open to various interpretations of the terroir. These tend towards a questionable level of objectivity because the fields they explore are not sufficient to explain the notion on their own, constituting only part of a whole.

Read More

Improvement of non-Saccharomyces yeast dominance during must fermentation by using spontaneous mutants resistant to SO2, EtOH and high pressure of CO2

AIM: A genetic study of four wine T. delbrueckii strains was done. Spore clones free of possible recessive growth‐retarding alleles with enhanced resistance to winemaking stressing conditions were obtained from these yeasts. METHODS: The genetic marker of resistance to cycloheximide (cyhR) allows easy monitoring of the new mutants obtained from these yeasts.

Read More

How sensor technologies combined with artificial intelligence increase the efficiency in grapevine breeding (research): current developments and future perspectives

Viticulture and grapevine breeding programs have to face and adapt to the rapidly changing growing conditions due to the ongoing climate change, the scarcity of resources and the demand for sustainability within the whole value chain of wine production. In times of highly effective and cost-efficient genotyping technologies routinely applied in plant research and breeding, the need for comparable high-speed and high-resolution phenotyping tools has increased substantially. The disciplines of grapevine research, breeding and precision viticulture picked up this demand – mostly independent from each other – by the development, validation and establishment of different sensor technologies in order to extend management strategies or to transform labor-intensive and expensive phenotyping.

Read More

Viticulture between adaptation and resilience: the role of the Italian long-term observatories for vineyard energy, water and carbon budgets

Viticulture is exposed to a range of new stressors, that are challenging its sustainability and disrupting famous and well-established production regions. Steady increase of average temperature, recurring heat waves, altered rainfall seasonal distribution, drought spells, increased pathogens pressure, they all mix up with increased frequency, making every growing season a special challenge and calling for new approaches to cope with worrying scenarios.

Read More

Effect of vine nitrogen status on grape and wine quality: Terroir study in the Vaud vineyard (Switzerland)

This study was conducted on soil-climate-plant relations (terroir) and their impact on grape composition and wine quality in the canton of Vaud by Agroscope Changins-Wädenswil ACW

Read More