title: Comprehensive selection of reference genes for quantitative gene expression analysis during seed development in Brassica napus.
abstract: MicroRNAs have higher expression stability than protein-coding genes in B. napus seeds and are therefore good reference genes for miRNA and mRNA RT-qPCR analysis. Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) has become the "gold standard" to gain insight into function of genes. However, the accuracy of the technique depends on appropriate reference genes for quantification analysis in different experimental conditions. Accumulation of microRNAs (miRNAs) has also been studied by RT-qPCR, but there are no reference genes currently validated for normalization of Brassica napus miRNA expression data. In this study, we selected 43 B. napus miRNAs and 18 previously validated mRNA reference genes. The expression stability of the candidate reference genes was evaluated in different tissue samples (stages of seed development, flowers, and leaves) using geNorm, NormFinder, and RefFinder analysis. The best-ranked reference genes for expression studies during seed development (miR167-1_2, miR11-1, miR159-1 and miR168-1) were used to asses the expression of miR03-1. Since candidate miRNAs showed higher expression stability than protein-coding genes in most of the tested conditions, the expression profile of DGAT1 gene was compared when normalized by the four most stable miRNAs reference genes and by the four most stable mRNA reference genes. The expected expression pattern of DGAT1 during seed development was achieved with the use of miRNA as reference genes. In conclusion, the most stable miRNA reference genes can be employed in the normalization of RT-qPCR quantification of miRNAs and protein-coding genes. This work is the first to perform a comprehensive survey of the stability of miRNA reference genes in B. napus and provides guidelines to obtain more accurate RT-qPCR results in B. napus seeds studies.
authors.affiliation: Departamento de Genética, PPGGBM, Universidade Federal do Rio Grande do Sul, UFRGS, Av. Bento Gonçalves 9500, Predio 43323 M, Sala 204/NGFP, Agronomia, Porto Alegre, RS, 91501-970, Brazil, dornelesronei@gmail.com.
authors.forename: Ronei Dorneles
authors.initials: RD
authors.lastname: Machado
authors.forename: Ana Paula
authors.initials: AP
authors.lastname: Christoff
authors.forename: Guilherme
authors.initials: G
authors.lastname: Loss-Morais
authors.forename: Márcia
authors.initials: M
authors.lastname: Margis-Pinheiro
authors.forename: Rogério
authors.initials: R
authors.lastname: Margis
authors.forename: Ana Paula
authors.initials: AP
authors.lastname: Körbes
mesh_terms: D029688:Brassica napus / Q000196:embryology* / Q000235:genetics*; D020869:Gene Expression Profiling; D018507:Gene Expression Regulation, Developmental; D018506:Gene Expression Regulation, Plant; D017343:Genes, Plant; D035683:MicroRNAs / Q000235:genetics / Q000378:metabolism; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction / Q000592:standards; D012015:Reference Standards; D015203:Reproducibility of Results; D012639:Seeds / Q000196:embryology* / Q000235:genetics*; D012984:Software
chemical_list: D035683:MicroRNAs; D010940:Plant Proteins; D012333:RNA, Messenger