Abstract
Maqui berry (Aristotelia chilensis) is a native Chilean species that produces berries that are exceptionally rich in anthocyanins and natural antioxidants. These natural compounds provide an array of health benefits for humans, making them very desirable in a fruit. At the same time, these substances also interfere with nucleic acid preparations, making RNA extraction from Maqui berry a major challenge. Our group established a method for RNA extraction of Maqui berry with a high quality RNA (good purity, good integrity and higher yield). This procedure is based on the adapted CTAB method using high concentrations of PVP (4 %) and β-mercaptoethanol (4 %) and spermidine in the extraction buffer. These reagents help to remove contaminants such as polysaccharides, proteins, phenols and also prevent the oxidation of phenolic compounds. The high quality of RNA isolated through this method allowed its uses with success in molecular applications for this endemic Chilean fruit, such as differential expression analysis of RNA-Seq data using next generation sequencing (NGS). Furthermore, we consider that our method could potentially be used for other plant species with extremely high levels of antioxidants and anthocyanins.
Highlights
RNA isolation has always been a critical first step in plant molecular biology
The samples obtained had no absorbance at A260 nm and they could not be visualized; it might be caused by the release of high contents of polyphenolics and metabolites after disruption of the cells, which are embedded in viscous polysaccharides during RNA extraction
We assayed various plant RNA extraction protocols for species and tissues with high levels of phenolic compounds and polysaccharides based on hexadecyltrimethyl ammonium bromide (CTAB), including the one developed by Jaakola et al 2001
Summary
RNA isolation has always been a critical first step in plant molecular biology. The structure of RNA contains a ribose sugars with a 2′-hydroxyl group attached that makes it more sensitive to degradation. This inherent instability together with the high levels of secondary metabolites found in plants hinders the RNA extraction and most of the times the RNA obtained has low concentration and poor quality (Sah et al 2014). Gene expression studies need to use a sufficient quantity of pure RNA to get accurate results (Yockteng et al 2013). Protocols aimed at the purification of copious amounts of high quality RNA are
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