Biomaterial Database

Validation of reference genes for the normalization of RT-qPCR gene expression in Acanthamoeba spp. 2020

Martina Köhsler, and David Leitsch, and Norbert Müller, and Julia Walochnik

Institute of Specific Prophylaxis und Tropical Medicine, Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna, Vienna, Austria.

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.

UI	MeSH Term	Description	Entries
D012015	Reference Standards	A basis of value established for the measure of quantity, weight, extent or quality, e.g. weight standards, standard solutions, methods, techniques, and procedures used in diagnosis and therapy.	Standard Preparations,Standards, Reference,Preparations, Standard,Standardization,Standards,Preparation, Standard,Reference Standard,Standard Preparation,Standard, Reference
D000048	Acanthamoeba	A genus of free-living soil amoebae that produces no flagellate stage. Its organisms are pathogens for several infections in humans and have been found in the eye, bone, brain, and respiratory tract.	Acanthamoebas
D000465	Algorithms	A procedure consisting of a sequence of algebraic formulas and/or logical steps to calculate or determine a given task.	Algorithm
D012337	RNA, Ribosomal, 18S	Constituent of the 40S subunit of eukaryotic ribosomes. 18S rRNA is involved in the initiation of polypeptide synthesis in eukaryotes.	18S Ribosomal RNA,18S RRNA,RNA, 18S Ribosomal,Ribosomal RNA, 18S
D060888	Real-Time Polymerase Chain Reaction	Methods used for detecting the amplified DNA products from the polymerase chain reaction as they accumulate instead of at the end of the reaction.	Kinetic Polymerase Chain Reaction,Quantitative Real-Time PCR,Quantitative Real-Time Polymerase Chain Reaction,Real-Time PCR,PCR, Quantitative Real-Time,PCR, Real-Time,PCRs, Quantitative Real-Time,PCRs, Real-Time,Quantitative Real Time PCR,Quantitative Real Time Polymerase Chain Reaction,Quantitative Real-Time PCRs,Real Time PCR,Real Time Polymerase Chain Reaction,Real-Time PCR, Quantitative,Real-Time PCRs,Real-Time PCRs, Quantitative
D020869	Gene Expression Profiling	The determination of the pattern of genes expressed at the level of GENETIC TRANSCRIPTION, under specific circumstances or in a specific cell.	Gene Expression Analysis,Gene Expression Pattern Analysis,Transcript Expression Analysis,Transcriptome Profiling,Transcriptomics,mRNA Differential Display,Gene Expression Monitoring,Transcriptome Analysis,Analyses, Gene Expression,Analyses, Transcript Expression,Analyses, Transcriptome,Analysis, Gene Expression,Analysis, Transcript Expression,Analysis, Transcriptome,Differential Display, mRNA,Differential Displays, mRNA,Expression Analyses, Gene,Expression Analysis, Gene,Gene Expression Analyses,Gene Expression Monitorings,Gene Expression Profilings,Monitoring, Gene Expression,Monitorings, Gene Expression,Profiling, Gene Expression,Profiling, Transcriptome,Profilings, Gene Expression,Profilings, Transcriptome,Transcript Expression Analyses,Transcriptome Analyses,Transcriptome Profilings,mRNA Differential Displays