title: Reference gene validation for quantitative RT-PCR during biotic and abiotic stresses in Vitis vinifera.
abstract: Grapevine is one of the most cultivated fruit crop worldwide with Vitis vinifera being the species with the highest economical importance. Being highly susceptible to fungal pathogens and increasingly affected by environmental factors, it has become an important agricultural research area, where gene expression analysis plays a fundamental role. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) is currently amongst the most powerful techniques to perform gene expression studies. Nevertheless, accurate gene expression quantification strongly relies on appropriate reference gene selection for sample normalization. Concerning V. vinifera, limited information still exists as for which genes are the most suitable to be used as reference under particular experimental conditions. In this work, seven candidate genes were investigated for their stability in grapevine samples referring to four distinct stresses (Erysiphe necator, wounding and UV-C irradiation in leaves and Phaeomoniella chlamydospora colonization in wood). The expression stability was evaluated using geNorm, NormFinder and BestKeeper. In all cases, full agreement was not observed for the three methods. To provide comprehensive rankings integrating the three different programs, for each treatment, a consensus ranking was created using a non-weighted unsupervised rank aggregation method. According to the last, the three most suitable reference genes to be used in grapevine leaves, regardless of the stress, are UBC, VAG and PEP. For the P. chlamydospora treatment, EF1, CYP and UBC were the best scoring genes. Acquaintance of the most suitable reference genes to be used in grapevine samples can contribute for accurate gene expression quantification in forthcoming studies.
authors.affiliation: Disease & Stress Biology Laboratory, Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Oeiras, Portugal; Disease & Stress Biology Laboratory, Instituto Superior de Agronomia, Universidade de Lisboa, Lisboa, Portugal.
authors.forename: Alexandre Filipe
authors.initials: AF
authors.lastname: Borges
authors.affiliation: Disease & Stress Biology Laboratory, Instituto Superior de Agronomia, Universidade de Lisboa, Lisboa, Portugal.
authors.forename: Catarina
authors.initials: C
authors.lastname: Fonseca
authors.affiliation: Disease & Stress Biology Laboratory, Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Oeiras, Portugal; Disease & Stress Biology Laboratory, Instituto Superior de Agronomia, Universidade de Lisboa, Lisboa, Portugal.
authors.forename: Ricardo Boavida
authors.initials: RB
authors.lastname: Ferreira
authors.affiliation: REQUIMTE, Departamento de Química, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, Caparica, Portugal.
authors.forename: Ana Maria
authors.initials: AM
authors.lastname: Lourenço
authors.affiliation: Disease & Stress Biology Laboratory, Instituto Superior de Agronomia, Universidade de Lisboa, Lisboa, Portugal.
authors.forename: Sara
authors.initials: S
authors.lastname: Monteiro
mesh_terms: D019295:Computational Biology; D020869:Gene Expression Profiling; D018506:Gene Expression Regulation, Plant; D017343:Genes, Plant; D009010:Monte Carlo Method; D018515:Plant Leaves / Q000235:genetics / Q000382:microbiology / Q000528:radiation effects; D060888:Real-Time Polymerase Chain Reaction; D013056:Spectrophotometry, Ultraviolet; D013312:Stress, Physiological; D014466:Ultraviolet Rays; D027843:Vitis / Q000235:genetics* / Q000382:microbiology* / Q000528:radiation effects