IBMC - Instituto de Biologia Molecular e Celular, Universidade do Porto, Porto, Portugal.
PLoS One. 2012;7(4):e34983. doi: 10.1371/journal.pone.0034983. Epub 2012 Apr 4.
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.
蓝藻是一群具有多样形态、最低营养需求和代谢可塑性的光合原核生物，这使它们成为生物技术应用中极具吸引力的生物。将这些生物体用作细胞工厂需要在系统水平上了解它们的生理学和代谢。对于基因转录本的定量，实时定量聚合酶链反应 (RT-qPCR) 是标准技术。然而，为了获得可靠的 RT-qPCR 结果，必须使用和验证参考基因。为此，我们从三种形态上明显不同的蓝藻中选择并分析了 12 个候选参考基因，这些蓝藻在常规使用的实验室条件下生长。在每个生物体中表现出较小变化的六个基因，使用 geNorm、NormFinder 和 BestKeeper 来评估它们的表达稳定性。此外，还确定了归一化所需的最少参考基因数。基于这三种算法，我们为蓝藻 RT-qPCR 数据归一化提供了一组基因列表。据我们所知，这是针对蓝藻参考基因验证的第一项工作，为未来的工作提供了有价值的起点。
