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.
Centre for Marine Bio-Innovation, Earth and Environmental Sciences, The University of New South Wales, Sydney, New South Wales, Australia; The School of Biotechnology and Biomolecular Sciences, Earth and Environmental Sciences, The University of New South Wales, Sydney, New South Wales, Australia.
Budoor
B
Alqarni
Centre for Marine Bio-Innovation, Earth and Environmental Sciences, The University of New South Wales, Sydney, New South Wales, Australia; The School of Biotechnology and Biomolecular Sciences, Earth and Environmental Sciences, The University of New South Wales, Sydney, New South Wales, Australia.
Brendan
B
Colley
University of Stuttgart, Institute of Technical Biochemistry, Allmandring 31, D-70569 Stuttgart, Germany.
Janosch
J
Klebensberger
The ithree Institute, The University of Technology Sydney, Australia; Singapore Centre for Environmental Life Sciences Engineering and the School of Biological Sciences, Nanyang Technological University, Singapore.
Diane
D
McDougald
Centre for Marine Bio-Innovation, Earth and Environmental Sciences, The University of New South Wales, Sydney, New South Wales, Australia; The School of Biological, Earth and Environmental Sciences, The University of New South Wales, Sydney, New South Wales, Australia; Singapore Centre for Environmental Life Sciences Engineering and the School of Biological Sciences, Nanyang Technological University, Singapore. Electronic address: RSCOTT@ntu.edu.sg.
Scott A
SA
Rice
D001426:Bacterial Proteins / Q000235:genetics*; D002244:Carbon / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D020043:Genes, Essential; D011550:Pseudomonas aeruginosa / Q000235:genetics* / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics; D011938:Rec A Recombinases / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*
D001426:Bacterial Proteins; D012333:RNA, Messenger; D002244:Carbon; D011938:Rec A Recombinases
Carbon starvation; Housekeeping genes normalisation; qRT-PCR