An improved method for RNA extraction from common bean seeds and validation of reference genes for qPCR.

• Autor(a) principal: PEREIRA, W. J.
• Data de Publicação: 2017
• Outros Autores: BASSINELLO, P. Z., BRONDANI, C., VIANELLO, R. P.
• Tipo de documento: Artigo
• Idioma: eng
• Título da fonte: Repositório Institucional da EMBRAPA (Repository Open Access to Scientific Information from EMBRAPA - Alice)
• Texto Completo: http://www.alice.cnptia.embrapa.br/alice/handle/doc/1076282
• topic: Gene normalization; Molecular breeding; Grain RNA extraction; Feijão; Phaseolus vulgaris; Melhoramento; RNA; Beans; Gene expression

An RNA extraction method with high integrity and purity as well as the selection of adequate reference genes are prerequisites for gene expression analysis. For common bean seeds, there is no well-defined protocol that can be used in a laboratory routine for gene expression analysis. In this study, an extraction protocol for RNA from common bean seeds, which produced material with good integrity for qPCR (RIN ≥ 6.5), was optimized. In addition, 10 reference genes were evaluated under qPCR standard conditions using different tissue samples of common beans. Gene stabilities were analyzed using the delta-CT method, Bestkeeper, NormFinder and geNorm approaches. The genes β-tubulin and T197 were ranked as the most stable among the sample sets evaluated with different tissue samples, while PvAct and Pv18S were the least stable. To our knowledge, this is the first study evaluating RNA isolation methods and reference gene selection for seeds of Phaseolus vulgaris.