Expression stability of 13 housekeeping genes during carbon starvation of Pseudomonas aeruginosa

Quantitative real-time polymerase chain reaction (qRT-PCR) is a reliable technique for quantifying mRNA levels when normalised by a stable reference gene/s. Many putative reference genes are known to be affected by physiological stresses, such as nutrient limitation and hence may not be suitable for normalisation. In this study of Pseudomonas aeruginosa, the expression of 13 commonly used reference genes, rpoS, proC, recA, rpsL, rho, oprL, anr, tipA, nadB, fabD, ampC, algD and gyrA, were analysed for changes in expression under carbon starvation and nutrient replete conditions. The results showed that rpoS was the only stably expressed housekeeping gene during carbon starvation. In contrast, other commonly used housekeeping genes were shown to vary by as much as 10–100 fold under starvation conditions. This study has identified a suitable reference gene for qRT-PCR in P. aeruginosa during carbon starvation. The results presented here highlight the need to validate housekeeping genes under the chosen experimental conditions. •Of thirteen putative housekeeping genes used previously for the normalisation of mRNA by qRT-PCR in Pseudomonas aeruginosa, only one gene was shown to be stable under carbon starvation conditions•The remaining putative housekeeping genes changed by 10–100 fold under starvation conditions•Housekeeping genes should be tested for stable expression under the particular conditions used, especially where physiological changes are being studied.