Fitoplancton Marino, S.L., El Puerto de Santa María, Cádiz, Spain.
Laboratory of Molecular Biology, Department of Biotechnology, Agricultural University of Athens, Athens, Greece.
PLoS One. 2021 Jan 14;16(1):e0245495. doi: 10.1371/journal.pone.0245495. eCollection 2021.
Quantitative real-time reverse transcription PCR (RT-qPCR) is a highly sensitive technique that can be applied to analyze how genes are modulated by culture conditions, but identification of appropriate reference genes for normalization is a critical factor to be considered. For this reason, the expression stability of 18 candidate reference genes was evaluated for the green microalgae Tetraselmis chui using the widely employed algorithms geNorm, NormFinder, BestKeeper, the comparative ΔCT method, and RefFinder. Microalgae samples were collected from large scale outdoor photobioreactors during the growing phase (OUT_GP), and during the semi-continuous phase at different times of the day (OUT_DC). Samples from standard indoor cultures under highly controlled conditions (IND) were also collected to complement the other data. Different rankings for the candidate reference genes were obtained depending on the culture conditions and the algorithm employed. After comparison of the achieved ranks with the different methods, the references genes selected for samples from specific culture conditions were ALD and EFL in OUT_GP, RPL32 and UBCE in OUT_DC, and cdkA and UBCE in IND. Moreover, the genes EFL and cdkA or EFL and UBCE appeared as appropriate combinations for pools generated from all samples (ALL). Examination in the OUT_DC cultures of genes encoding the large and small subunits of ADP-glucose pyrophosphorylase (AGPL and AGPS, respectively) confirmed the reliability of the identified reference genes, RPL32 and UBCE. The present study represents a useful contribution for studies of gene expression in T. chui, and also represents the first step to set-up an RT-qPCR platform for quality control of T. chui biomass production in industrial facilities.
实时荧光定量逆转录聚合酶链反应（RT-qPCR）是一种高度敏感的技术，可用于分析基因如何受培养条件调控，但鉴定合适的内参基因进行标准化是一个关键因素。出于这个原因，使用广泛应用的 geNorm、NormFinder、BestKeeper、比较 ΔCT 方法和 RefFinder 算法评估了钝顶螺旋藻的 18 个候选内参基因的表达稳定性。微藻样品是在生长阶段（OUT_GP）从大型户外光生物反应器中收集的，以及在不同时间的半连续阶段（OUT_DC）从大型户外光生物反应器中收集的。还收集了在高度受控条件下进行的标准室内培养（IND）的样品，以补充其他数据。根据培养条件和所使用的算法，候选内参基因的排名不同。将获得的排名与不同方法进行比较后，为特定培养条件的样品选择的参考基因是 OUT_GP 中的 ALD 和 EFL、OUT_DC 中的 RPL32 和 UBCE，以及 IND 中的 cdkA 和 UBCE。此外，EFL 和 cdkA 或 EFL 和 UBCE 这两个基因似乎是所有样品（ALL）生成的池的合适组合。在 OUT_DC 培养物中对 ADP-葡萄糖焦磷酸化酶大亚基和小亚基编码基因（AGPL 和 AGPS）的检查证实了鉴定的内参基因 RPL32 和 UBCE 的可靠性。本研究为钝顶螺旋藻基因表达研究提供了有用的贡献，也是为工业设施中钝顶螺旋藻生物量生产建立 RT-qPCR 质量控制平台的第一步。
