title: RNA isolation from loquat and other recalcitrant woody plants with high quality and yield.
abstract: RNA isolation is difficult in plants that contain large amounts of polysaccharides and polyphenol compounds. To date, no commercial kit has been developed for the isolation of high-quality RNA from tissues with these characteristics, especially for fruit. The common protocols for RNA isolation are tedious and usually result in poor yields when applied to recalcitrant plant tissues. Here an efficient RNA isolation protocol based on cetyltrimethylammonium bromide (CTAB) and two successive precipitations with 10 M lithium chloride (LiCl) was developed specifically for loquat fruits, but it was proved to work efficiently in other tissues of loquat and woody plants. The RNA isolated by this improved protocol was not only of high purity and integrity (A260/A280 ratios ranged from 1.90 to 2.04 and A260/A230 ratios were>2.0) but also of high yield (up to 720 μg on average [coefficient of variation=21%] total RNA per gram fresh tissue). The protocol was tested on loquat fruit (different stages of development, postharvest, ripening, and bruising), leaf, root, flower, stem, and bud; quince fruit and root; grapevine cells in liquid culture; and rose petals. The RNA obtained with this method is amenable to enzymatic treatments and can be efficiently applied for research on gene characterization, expression, and function.
authors.affiliation: Plant Proteomics and Functional Genomics Group, Department of Agrochemistry and Biochemistry, Faculty of Science, University of Alicante, 03690 San Vicente del Raspeig, Alicante, Spain; Biotechnology and Molecular Biology Group, Quevedo State Technical University, EC-120501 Quevedo, Ecuador.
authors.forename: Jaime
authors.initials: J
authors.lastname: Morante-Carriel
authors.affiliation: Research Technical Facility, Proteomics and Genomics Division, University of Alicante, 03690 San Vicente del Raspeig, Alicante, Spain.
authors.forename: Susana
authors.initials: S
authors.lastname: Sellés-Marchart
authors.affiliation: Plant Proteomics and Functional Genomics Group, Department of Agrochemistry and Biochemistry, Faculty of Science, University of Alicante, 03690 San Vicente del Raspeig, Alicante, Spain.
authors.forename: Ascensión
authors.initials: A
authors.lastname: Martínez-Márquez
authors.affiliation: Plant Proteomics and Functional Genomics Group, Department of Agrochemistry and Biochemistry, Faculty of Science, University of Alicante, 03690 San Vicente del Raspeig, Alicante, Spain.
authors.forename: María José
authors.initials: MJ
authors.lastname: Martínez-Esteso
authors.affiliation: Instituto de Bioquímica Vegetal y Fotosíntesis, CSIC and Universidad de Sevilla, 41092 Seville, Spain.
authors.forename: Ignacio
authors.initials: I
authors.lastname: Luque
authors.affiliation: Plant Proteomics and Functional Genomics Group, Department of Agrochemistry and Biochemistry, Faculty of Science, University of Alicante, 03690 San Vicente del Raspeig, Alicante, Spain. Electronic address: roque.bru@ua.es.
authors.forename: Roque
authors.initials: R
authors.lastname: Bru-Martínez
mesh_terms: D000077286:Cetrimonium; D002593:Cetrimonium Compounds / Q000737:chemistry; D005591:Chemical Fractionation / Q000379:methods*; D031983:Eriobotrya / Q000737:chemistry* / Q000235:genetics / Q000254:growth & development; D018745:Genome, Plant / Q000235:genetics; D018749:RNA, Plant / Q000737:chemistry / Q000302:isolation & purification*; D014934:Wood / Q000737:chemistry* / Q000235:genetics / Q000254:growth & development
chemical_list: D002593:Cetrimonium Compounds; D018749:RNA, Plant; D000077286:Cetrimonium
keywords: Fruits; Polyphenols; Polysaccharides; RNA isolation; Woody plants