Prencipe Annalisa, Salerno Antonella, D'Amico Margherita, Marsico Antonio Domenico, Ventura Mario, Velasco Riccardo, Cardone Maria Francesca, Bergamini Carlo, Maggiolini Flavia Angela Maria
Department of Biosciences, Biotechnology and Environment, University of Bari "Aldo Moro", 70125 Bari, Italy.
Council for Agricultural Research and Economics-Research Center Viticulture and Enology (CREA-VE), Via Casamassima 148, 70010 Turi, Italy.
Plants (Basel). 2025 Mar 21;14(7):988. doi: 10.3390/plants14070988.
Extracting high-quality RNA from grape () berry skins is challenging due to their high levels of polysaccharides, phenolic compounds, sugars, and organic acids, which can negatively impact RNA purity and yield. Indeed, polyphenols can bind to RNA, polysaccharides may co-precipitate, and sugars and organic acids can interfere with the pH and ionic properties of the extraction buffer. Commercial kits offer a quick extraction method but are often ineffective for grape berry skins. Similarly, protocols that work well for other vegetal tissues are also inefficient and time-consuming for this tissue. To overcome these limitations, we optimized the RNA isolation by adding a sorbitol pre-wash step to both a non-commercial protocol and a commercial kit. Our results show that it significantly improves the RNA yield and quality from grape berry skins, increasing the RNA purity and integrity, as evidenced by higher RIN (RNA Integrity Number) values and better Qubit and Nanodrop measurements. The strategy's efficacy was further validated through RNA sequencing, yielding high-quality reads with low error rates, suitable for gene expression studies. Thus, incorporating a sorbitol pre-wash step improves the RNA yield and quality from grape berry skins making it suitable for high-throughput sequencing, and provides a reliable tool for advancing grapevine research.
从葡萄浆果表皮中提取高质量RNA具有挑战性,因为其富含多糖、酚类化合物、糖类和有机酸,这些物质会对RNA的纯度和产量产生负面影响。事实上,多酚会与RNA结合,多糖可能会共沉淀,糖类和有机酸会干扰提取缓冲液的pH值和离子特性。商业试剂盒提供了一种快速提取方法,但对葡萄浆果表皮往往无效。同样,对其他植物组织有效的方案对这种组织也效率低下且耗时。为了克服这些限制,我们通过在非商业方案和商业试剂盒中都增加一个山梨醇预洗涤步骤来优化RNA分离。我们的结果表明,这显著提高了葡萄浆果表皮的RNA产量和质量,提高了RNA的纯度和完整性,更高的RIN(RNA完整性数值)值以及更好的Qubit和Nanodrop测量结果证明了这一点。通过RNA测序进一步验证了该策略的有效性,产生了低错误率的高质量读数,适用于基因表达研究。因此,加入山梨醇预洗涤步骤提高了葡萄浆果表皮的RNA产量和质量,使其适用于高通量测序,并为推进葡萄研究提供了一个可靠的工具。