title: Validation of novel reference genes for reverse transcription quantitative real-time PCR in drought-stressed sugarcane.
abstract: One of the most challenging aspects of RT-qPCR data analysis is the identification of reliable reference genes. Ideally, they should be neither induced nor repressed under different experimental conditions. To date, few reference genes have been adequately studied for sugarcane (Saccharum spp.) using statistical approaches. In this work, six candidate genes ( αTUB, GAPDH, H1, SAMDC, UBQ, and 25S rRNA) were tested for gene expression normalization of sugarcane root tissues from drought-tolerant and -sensitive accessions after continuous dehydration (24 h). By undergoing different approaches (GeNorm, NormFinder, and BestKeeper), it was shown that most of them could be used in combinations for normalization purposes, with the exception of SAMDC. Nevertheless three of them (H1, αTUB, and GAPDH) were considered the most reliable reference genes. Their suitability as reference genes validated the expression profiles of two targets (AS and PFP α1), related to SuperSAGE unitags, in agreement with results revealed by previous in silico analysis. The other two sugarcane unitags (ACC oxidase and PIP1-1), after salt stress (100 mM NaCl), presented their expressions validated in the same way. In conclusion, these reference genes will be useful for dissecting gene expression in sugarcane roots under abiotic stress, especially in transcriptomic studies using SuperSAGE or RNAseq approaches.
authors.affiliation: Federal University of Pernambuco (UFPE/CCB/Genética), 50670-420 Recife, PE, Brazil.
authors.forename: Roberta Lane de Oliveira
authors.initials: RL
authors.lastname: Silva
authors.affiliation: Federal University of Pernambuco (UFPE/CCB/Genética), 50670-420 Recife, PE, Brazil.
authors.forename: Manassés Daniel
authors.initials: MD
authors.lastname: Silva
authors.affiliation: Federal University of Pernambuco (UFPE/CCB/Genética), 50670-420 Recife, PE, Brazil.
authors.forename: José Ribamar Costa
authors.initials: JR
authors.lastname: Ferreira Neto
authors.affiliation: ABC Medical School, 09060-650 São Paulo, SP, Brazil.
authors.forename: Claudia Huerta
authors.initials: CH
authors.lastname: de Nardi
authors.affiliation: Sugarcane Technology Center (CTC), 13400-970 Piracicaba, SP, Brazil.
authors.forename: Sabrina Moutinho
authors.initials: SM
authors.lastname: Chabregas
authors.affiliation: Sugarcane Technology Center (CTC), 13400-970 Piracicaba, SP, Brazil.
authors.forename: William Lee
authors.initials: WL
authors.lastname: Burnquist
authors.affiliation: Institute of Molecular Biosciences, Goethe University Frankfurt am Main, 60438 Frankfurt am Main, Germany.
authors.forename: Günter
authors.initials: G
authors.lastname: Kahl
authors.affiliation: Federal University of Pernambuco (UFPE/CCB/Genética), 50670-420 Recife, PE, Brazil.
authors.forename: Ana Maria
authors.initials: AM
authors.lastname: Benko-Iseppon
authors.affiliation: Federal University of Pernambuco (UFPE/CCB/Genética), 50670-420 Recife, PE, Brazil.
authors.forename: Ederson Akio
authors.initials: EA
authors.lastname: Kido
mesh_terms: D019295:Computational Biology; D055864:Droughts; D018506:Gene Expression Regulation, Plant; D017343:Genes, Plant; D060888:Real-Time Polymerase Chain Reaction; D015203:Reproducibility of Results; D031786:Saccharum / Q000235:genetics* / Q000378:metabolism; D012680:Sensitivity and Specificity; D013312:Stress, Physiological / Q000235:genetics*