title: Selection of suitable reference genes for RT-qPCR analyses in cyanobacteria.
abstract: Cyanobacteria are a group of photosynthetic prokaryotes that have a diverse morphology, minimal nutritional requirements and metabolic plasticity that has made them attractive organisms to use in biotechnological applications. The use of these organisms as cell factories requires the knowledge of their physiology and metabolism at a systems level. For the quantification of gene transcripts real-time quantitative polymerase chain reaction (RT-qPCR) is the standard technique. However, to obtain reliable RT-qPCR results the use and validation of reference genes is mandatory. Towards this goal we have selected and analyzed twelve candidate reference genes from three morphologically distinct cyanobacteria grown under routinely used laboratory conditions. The six genes exhibiting less variation in each organism were evaluated in terms of their expression stability using geNorm, NormFinder and BestKeeper. In addition, the minimum number of reference genes required for normalization was determined. Based on the three algorithms, we provide a list of genes for cyanobacterial RT-qPCR data normalization. To our knowledge, this is the first work on the validation of reference genes for cyanobacteria constituting a valuable starting point for future works.
authors.affiliation: IBMC - Instituto de Biologia Molecular e Celular, Universidade do Porto, Porto, Portugal.
authors.forename: Filipe
authors.initials: F
authors.lastname: Pinto
authors.forename: Catarina C
authors.initials: CC
authors.lastname: Pacheco
authors.forename: Daniela
authors.initials: D
authors.lastname: Ferreira
authors.forename: Pedro
authors.initials: P
authors.lastname: Moradas-Ferreira
authors.forename: Paula
authors.initials: P
authors.lastname: Tamagnini
mesh_terms: D000465:Algorithms; D000458:Cyanobacteria / Q000235:genetics* / Q000302:isolation & purification*; D020869:Gene Expression Profiling; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012015:Reference Standards