Abstract
Acanthamoebae are potentially pathogenic organisms, with a highly unique, yet still insufficiently investigated metabolism. Many open questions can be addressed by gene expression studies, however, for Acanthamoeba reliable standards have not yet been established. In this study, suitable reference genes (RGs) for RT-qPCR in Acanthamoeba were comprehensively evaluated, comparing different Acanthamoeba strains and employing four different algorithms (NormFinder, GeNorm, BestKeeper and RefFinder). Expression stability was assessed under various conditions and the potentials of the most promising RGs for accurate normalization of target genes were evaluated. Expression stability of RGs varied depending on conditions and employed algorithms, however, the genes for the 18S rRNA and the hypoxanthine phosphoribosyl transferase seem to be widely suitable RGs. Normalization with a combination of two carefully chosen RGs resulted in reliable expression data for target genes, while normalization with unsuitable RGs led to significant misinterpretation of expression profiles. Thus, a careful evaluation of RGs prior to expression studies is essential.
Highlights
Acanthamoebae are potentially pathogenic organisms, with a highly unique, yet still insufficiently investigated metabolism
The initial selection was based on RT-qPCR efficiency and dispersion of quantification cycle (Cq) values
A reliable quantification of gene expression mainly depends on an accurate normalization
Summary
Acanthamoebae are potentially pathogenic organisms, with a highly unique, yet still insufficiently investigated metabolism. Under certain circumstances they can act as facultative pathogens being the causative agents of Acanthamoeba keratitis, an often seriously progressing keratitis, occurring predominantly in contact lens wearers They can cause several disseminating infections, mostly in immunocompromised individuals, potentially resulting in granulomatous amoebic encephalitis (GAE). The best and most common method utilized for the analysis of relative changes in the mRNA expression of target genes is the parallel quantification of standard reference genes (RGs), which ideally are constitutively expressed www.nature.com/scientificreports housekeeping genes, required for the maintenance of basic cellular functions at all time[3] This strategy eliminates variation introduced by pre-PCR and PCR processing. A careful evaluation of the employed RGs prior to large experiments is advisable in addition to following guidelines established for quantitative real-time PCR7
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