Reference genes for real-time RT-PCR expression studies in an Antarctic Pseudomonas exposed to different temperature conditions
Psychrophilic and psychrotolerant bacteria from permanently cold environments may be the most abundant extremophiles on Earth and yet little is known on how they cope with temperature stress. Real-time reverse transcription PCR (RT-qPCR) is a powerful technique that could shed light on this matter but it requires pre-validated reference genes for normalization of data to get accurate results. In this study, we assessed the expression stability of eight candidate genes for the psychrotolerant Antarctic isolate Pseudomonas sp. AU10 during exponential growth under 4 °C and 30 °C, and after a cold-shock. Using the software programs BestKeeper and geNorm we validated recA, ftsZ, 16S rRNA, and rpoD as reference genes and we suggested the combination of recA and ftsZ for qPCR data normalization. Our results provide a starting point for gene expression studies in Antarctic Pseudomonas concerning temperature-related physiology and also for the validation of reference genes in other cold-adapted bacterial species.
KeywordsReference genes Validation RT-qPCR Pseudomonas Psychrotolerant Cold-shock
The authors especially thank Prof. Lucia Yim and MSc Mailén Arleo for their support and valuable suggestions during this work. They also thank the Uruguayan Antarctic Institute for the logistic support during the stay in the Antarctic Base Artigas. A. Ramón and S. Castro-Sowinski are members of the National Research System (SNI, Sistema Nacional de Investigadores). This work was partially supported by PEDECIBA (Programa de Desarrollo de las Ciencias Básicas) and CSIC (Comisión Sectorial de Investigación Científica, C335-348). The work of CXGL was supported by ANII (Agencia Nacional de Investigación e Innovación) and CAP (Comisión Académica de Posgrado).
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