title: Microarray-based uncovering reference genes for quantitative real time PCR in grapevine under abiotic stress.
abstract: BACKGROUND
  Quantitative real time polymerase chain reaction is becoming the primary tool for detecting mRNA and transcription data analysis as it shows to have advantages over other more commonly used techniques. Nevertheless, it also presents a few shortcomings, with the most import being the need for data normalisation, usually with a reference gene. Therefore the choice of the reference gene(s) is of great importance for correct data analysis. Microarray data, when available, can be of great assistance when choosing reference genes. Grapevine was submitted to water stress and heat stress as well as a combination of both to test the stability of the possible reference genes.
  
  
  RESULTS
  Using the analysis of microarray data available for grapevine, six possible reference genes were selected for RT-qPCR validation: PADCP, ubiq, TIF, TIF-GTP, VH1-IK, aladin-related. Two additional genes that are commonly used as reference genes were included: act and L2. The stability of those genes was tested in leaves of grapevine in both field plants and in greenhouse plants under water or heat stress or a combination of both. Gene stability was analyzed with the softwares GeNorm, NormFinder and the ΔCq method resulting in several combinations of reference genes suitable for data normalisation. In order to assess the best combination, the reference genes were tested in putative stress marker genes (PCO, Galsynt, BKCoAS and HSP17) also chosen from the same microarray, in water stress, heat stress and the combination of both.
  
  
  CONCLUSIONS
  Each method selected different gene combinations (PADCP + act, TIF + TIF-GTP and ubiq + act). However, as none of the combinations diverged significantly from the others used to normalize the expression of the putative stress marker genes, then any combination is suitable for data normalisation under the conditions tested. Here we prove the accuracy of choosing grapevine reference genes for RT-qPCR through a microarray analysis.
authors.affiliation: Centro de Botânica Aplicado à Agricultura, Departamento de Recursos Naturais, Ambiente e Território, Instituto Superior de Agronomia, Universidade Técnica de Lisboa, Lisbon, Portugal.
authors.forename: João L
authors.initials: JL
authors.lastname: Coito
authors.forename: Margarida
authors.initials: M
authors.lastname: Rocheta
authors.forename: Luísa
authors.initials: L
authors.lastname: Carvalho
authors.forename: Sara
authors.initials: S
authors.lastname: Amâncio
mesh_terms: D002138:Calibration; D019295:Computational Biology; D030541:Databases, Genetic; D020869:Gene Expression Profiling / Q000379:methods*; D018506:Gene Expression Regulation, Plant; D005819:Genetic Markers; D018869:Heat-Shock Response / Q000235:genetics; D020411:Oligonucleotide Array Sequence Analysis; D018515:Plant Leaves / Q000378:metabolism; D020871:RNA Stability; D012333:RNA, Messenger / Q000378:metabolism*; D018749:RNA, Plant / Q000378:metabolism*; D060888:Real-Time Polymerase Chain Reaction / Q000592:standards*; D012015:Reference Standards; D015203:Reproducibility of Results; D013312:Stress, Physiological / Q000235:genetics*; D027843:Vitis / Q000235:genetics* / Q000378:metabolism
chemical_list: D005819:Genetic Markers; D012333:RNA, Messenger; D018749:RNA, Plant