Validation of reference genes for the normalization of RT-qPCR gene expression in Acanthamoeba spp.

Köhsler, Martina; Leitsch, David; Müller, Norbert; Walochnik, Julia (2020). Validation of reference genes for the normalization of RT-qPCR gene expression in Acanthamoeba spp. Scientific Reports, 10(1), p. 10362. Nature Publishing Group 10.1038/s41598-020-67035-0

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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.

Item Type:

Journal Article (Original Article)

Division/Institute:

04 Faculty of Medicine > Service Sector > Institute for Infectious Diseases > Parasitology
04 Faculty of Medicine > Service Sector > Institute for Infectious Diseases
05 Veterinary Medicine > Department of Infectious Diseases and Pathobiology (DIP) > Institute of Parasitology
05 Veterinary Medicine > Department of Infectious Diseases and Pathobiology (DIP)

UniBE Contributor:

Müller, Norbert

Subjects:

500 Science > 570 Life sciences; biology
600 Technology > 610 Medicine & health
600 Technology > 630 Agriculture

ISSN:

2045-2322

Publisher:

Nature Publishing Group

Language:

English

Submitter:

Achim Braun Parham

Date Deposited:

17 Mar 2021 10:34

Last Modified:

17 Mar 2021 16:12

Publisher DOI:

10.1038/s41598-020-67035-0

PubMed ID:

32587282

BORIS DOI:

10.48350/154117

URI:

https://boris.unibe.ch/id/eprint/154117

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