Selection of reliable reference genes for RT-qPCR analysis during developmental stages and abiotic stress in Setaria viridis.
Real-time PCR (RT-qPCR) expression analysis is a powerful analytical technique, but reliable results depend on the use of stable reference genes for proper normalization. This study proposed to test the expression stability of 13 candidate reference genes in Setaria viridis, a monocot species recently proposed as a new C4 model plant. Gene expression stability of these genes was assayed across different tissues and developmental stages of Setaria and under drought or aluminum stress. In general, our results showed Protein Kinase, RNA Binding Protein and SDH as the most stable genes. Moreover, pairwise analysis showed that two reference genes were sufficient to normalize the gene expression data under each condition. By contrast, GAPDH and ACT were the least stably expressed genes tested. Validation of suitable reference genes was carried out to profile the expression of P5CS and GolS during abiotic stress. In addition, normalization of gene expression of SuSy, involved in sugar metabolism, was assayed in the developmental dataset. This study provides a list of reliable reference genes for transcript normalization in S. viridis in different tissues and stages of development and under abiotic stresses, which will facilitate genetic studies in this monocot model plant.
Genetics and Biotechnology Laboratory, Embrapa Agroenergy (CNPAE), Brasília, DF, 70770-901, Brazil.
Polyana Kelly
PK
Martins
Brazilian Bioethanol Science and Technology Laboratory/Brazilian Center of Research in Energy and Materials, Campinas, SP, 13083-100, Brazil.
Valéria
V
Mafra
Genetics and Biotechnology Laboratory, Embrapa Agroenergy (CNPAE), Brasília, DF, 70770-901, Brazil.
Wagner Rodrigo
WR
de Souza
Genetics and Biotechnology Laboratory, Embrapa Agroenergy (CNPAE), Brasília, DF, 70770-901, Brazil.
Ana Paula
AP
Ribeiro
Genetics and Biotechnology Laboratory, Embrapa Agroenergy (CNPAE), Brasília, DF, 70770-901, Brazil.
Felipe
F
Vinecky
Genetics and Biotechnology Laboratory, Embrapa Agroenergy (CNPAE), Brasília, DF, 70770-901, Brazil.
Marcos Fernando
MF
Basso
Genetics and Biotechnology Laboratory, Embrapa Agroenergy (CNPAE), Brasília, DF, 70770-901, Brazil.
Bárbara Andrade Dias Brito
BA
da Cunha
Genetics and Biotechnology Laboratory, Embrapa Agroenergy (CNPAE), Brasília, DF, 70770-901, Brazil.
Adilson Kenji
AK
Kobayashi
Genetics and Biotechnology Laboratory, Embrapa Agroenergy (CNPAE), Brasília, DF, 70770-901, Brazil.
Hugo Bruno Correa
HB
Molinari
D000465:Algorithms; D000535:Aluminum / Q000737:chemistry; D055864:Droughts; D020869:Gene Expression Profiling; D018507:Gene Expression Regulation, Developmental; D018506:Gene Expression Regulation, Plant; D017343:Genes, Plant; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction; D032983:Setaria Plant / Q000235:genetics* / Q000378:metabolism*; D013312:Stress, Physiological / Q000235:genetics*
D010940:Plant Proteins; D000535:Aluminum