Isolation of high-quality rna from grains of different maize varieties

• Published in: Preparative biochemistry & biotechnology 2014-10, Vol.44 (7), p.697-707
• Main Authors: Messias, Rafael da Silva, Galli, Vanessa, Buss, Julieti Huch, Borowski, Joyce Moura, Nora, Leonardo, e Silva, Sérgio Delmar dos Anjos, Margis, Rogério, Rombaldi, Cesar Valmor
• Format: Article
• Language: English
• Subjects: Biochemistry; Biochemistry - methods; carotenoid biosynthesis; Cetrimonium Compounds - chemistry; Corn; Deoxyribonucleic acid; DNA; Gene expression; Mixed Function Oxygenases - genetics; Polymerase chain reaction; Real-Time Polymerase Chain Reaction; Ribonucleic acid; RNA; RNA extraction; RNA, Plant - isolation & purification; Seeds - genetics; Zea mays; Zea mays - genetics; Zea mays L
• ISSN: 1532-2297; 1082-6068; 1532-2297
• DOI: 10.1080/10826068.2013.868355

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.