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Background  Quantitative real-time PCR is a valuable tool for evaluating bacterial gene expression. However, in order to make best use of this method, endogenous reference genes for expression data normalisation must first be identified by carefully validating the stability of expression under experimental conditions. Therefore, the objective of this study was to validate eight reference genes of the opportunistic swine pathogen,  Actinobacillus suis , grown in aerobic cultures with (Epinephrine) or without (Aerobic) epinephrine in the growth medium and in anoxic static cultures (Anoxic), and sampled during exponential and stationary phases.    Results  Using the RefFinder tool, expression data were analysed to determine whether comprehensive stability rankings of selected reference genes varied with experimental design. When comparing Aerobic and Epinephrine cultures by growth phase,  pyk  and  rpoB  were both among the most stably expressed genes, but when analysing both growth phases together, only  pyk  remained in the top three rankings. When comparing Aerobic and Anoxic samples,  proS  ranked among the most stable genes in exponential and stationary phase data sets as well as in combined rankings. When analysing the Aerobic, Epinephrine, and Anoxic samples together, only  gyrA  ranked consistently among the top three most stably expressed genes during exponential and stationary growth as well as in combined rankings; the  rho  gene ranked as least stably expressed gene in this data set.    Conclusions  Reference gene stability should be carefully assessed with the design of the experiment in mind. In this study, even the commonly used reference gene  16S rRNA  demonstrated large variability in stability depending on the conditions studied and how the data were analysed. As previously suggested, the best approach may be to use a geometric mean of multiple genes to normalise qPCR results. As researchers continue to validate reference genes for various organisms in multiple growth conditions and sampling time points, it may be possible to make informed predictions as to which genes may be most suitable to validate for a given experimental design, but in the meantime, the reference genes used to normalise qPCR data should be selected with caution.

Validation of reference genes for quantitative real-time PCR (qPCR) analysis of Actinobacillus suis