Abstract
BackgroundQuantitative 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.ResultsUsing 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.ConclusionsReference 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.
Highlights
Quantitative real-time PCR is a valuable tool for evaluating bacterial gene expression
Growth curves to determine sampling time points To determine the times of early exponential and early stationary growth phase, growth curves of aerobic cultures with (Epinephrine) or without (Aerobic) 50 μM epinephrine added to the growth medium at the time of inoculation, and anoxic static cultures (Anoxic) (Figure 1) were done
To control for sample variability [14], once the exponential and early stationary growth phase time points were identified, the volume of culture sampled was adjusted to ensure that an approximately equal number of cells was collected for RNA extraction each time; cell numbers were enumerated by plate counting
Summary
Quantitative real-time PCR is a valuable tool for evaluating bacterial gene expression. 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. Actinobacillus suis is a Gram negative facultative anaerobe which is a frequent member of the normal microbiome of swine tonsils of the soft palate [1]. It is an important pathogen in pigs of all ages, where it can cause septicaemia and sequelae such as meningitis, arthritis, and pleuropneumonia [2]. Quantitative real-time PCR (qPCR) is a sensitive method for the determination of bacterial gene expression. It has been suggested that using the geometric mean of data collected from multiple reference genes is more appropriate than relying on a single reference gene for normalisation [7]
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