title: Isolation of high-quality RNA from grains of different maize varieties.
abstract: The study of gene expression in maize varieties represents a powerful tool aiming to increase vitamin A precursors. However, the isolation of RNA from different maize varieties is challenging because these varieties show different levels of polysaccharides, and most methods available for RNA isolation are inappropriate for grain samples. The polysaccharides co-purify and co-precipitate with RNA during isolation, resulting in low-quality RNA, compromising the use of RNA in subsequent applications. Thus, a cetyltrimethylammonium bromide (CTAB)-based method was adapted in this study and compared with six methods for RNA isolation, including commercial reagents and RNA and DNA isolation kits, in order to identify the most appropriate for maize grains from different varieties. Most of the methods evaluated were considered inadequate due to limitations in terms of purity and/or quantity of the isolated RNA, which affected the efficiency of subsequent RT-qPCR analysis, resulting in nonamplification of β-carotene hydroxylase gene (HYD3) or high deviation among replicates. However, the CTAB modified method allowed the study to obtain intact RNA, with high quality and quantity, from 25 maize varieties. Furthermore, this RNA was successfully used to evaluate the expression of HYD3 gene by real-time qualitative polymerase chain reaction (RT-qPCR), and thus represents a simple, efficient, and low-cost strategy.
authors.affiliation: a Embrapa Clima Temperado , Pelotas , Rio Grande do Sul , Brasil.
authors.forename: Rafael da Silva
authors.initials: Rda S
authors.lastname: Messias
authors.forename: Vanessa
authors.initials: V
authors.lastname: Galli
authors.forename: Julieti Huch
authors.initials: JH
authors.lastname: Buss
authors.forename: Joyce Moura
authors.initials: JM
authors.lastname: Borowski
authors.forename: Leonardo
authors.initials: L
authors.lastname: Nora
authors.forename: Sérgio Delmar dos Anjos
authors.initials: SD
authors.lastname: e Silva
authors.forename: Rogério
authors.initials: R
authors.lastname: Margis
authors.forename: Cesar Valmor
authors.initials: CV
authors.lastname: Rombaldi
mesh_terms: D001671:Biochemistry / Q000379:methods*; D000077286:Cetrimonium; D002593:Cetrimonium Compounds / Q000737:chemistry; D006899:Mixed Function Oxygenases / Q000235:genetics; D018749:RNA, Plant / Q000302:isolation & purification*; D060888:Real-Time Polymerase Chain Reaction; D012639:Seeds / Q000235:genetics; D003313:Zea mays / Q000235:genetics*
chemical_list: D002593:Cetrimonium Compounds; D018749:RNA, Plant; D006899:Mixed Function Oxygenases; C079232:beta-carotene hydroxylase; D000077286:Cetrimonium
keywords: RNA extraction; Zea mays L.; carotenoid biosynthesis; gene expression