ArticleBMC research notes2015
Validation of reference genes for quantitative real-time PCR (qPCR) analysis of Actinobacillus suis.
Article in BMC research notes, 2015. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 3 papers.
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Who cites it
3 citing papers in PubMed.
- The carbon source-dependent pattern of antimicrobial activity and gene expression in Pseudomonas donghuensis P482.Scientific reports · 2021Article
- Attachment of Actinobacillus suis H91-0380 and Its Isogenic Adhesin Mutants to Extracellular Matrix Components of the Tonsils of the Soft Palate of Swine.Infection and immunity · 2016Article
- Validation of Reference Genes for Real-Time Quantitative PCR (qPCR) Analysis of Avibacterium paragallinarum.PloS one · 2016Article
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2 authors.
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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.
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