Abstract
The bank vole has become an important species for the study of gene expression changes underlying evolutionary adaptation, within-host dynamics and resistance to pathogens, or response to pollutants. RT–qPCR is an optimized method for the rapid and accurate measuring gene expression, but it relies on the use of reference genes (RGs) as endogenous controls to normalize mRNA levels of the target genes. Contrary to the common practice, the expression level of the housekeeping genes traditionally used as RGs cannot be assumed stable across different species and experimental settings. Furthermore, compared to model laboratory species, there are potentially additional sources of variation when collecting gene expression data from natural populations with unknown genetic and environmental backgrounds. Thus, rigorous determination of RGs in natural populations of the bank vole was necessary to facilitate gene expression studies in this emerging model species. Therefore, we evaluated the expression variation of 10 RG candidates in spleen samples of bank voles spanning a broad latitudinal range across Europe, using four approaches. Ube2d2a, Ppia and Tbp were consistently identified as the least variable genes, followed by Rn18s, Ywhaz and Rplp0. The combinations Ube2d2a and Ppia or Ube2d2a and Tbp were identified to be sufficient for the normalization. In contrast, the traditional housekeeping genes Actb, Gapdh, Sdha and Tuba1a displayed large expression variation and are not recommended as internal controls. Our results underscore the need of a systematic evaluation of appropriate reference genes to each particular experimental system and provide a useful starting point for further studies.
Highlights
Regulatory variation in gene expression is increasingly being recognized as a major determinant of phenotypic variation (Lai et al, 2008; Fraser, 2013; Sandkam et al, 2015; Lin et al, 2017)
Studies and represented the three latitudinal zones identified based on the combined information from phylogenetic analyses of mitochondrial DNA sequences (Filipi et al, 2015) and clustering of genomic data (Marková et al, 2020): southern refugial (n = 6), which includes the areas of glacial refugia in the Mediterranean region, northern apex (n = 7), which includes Great Britain and Fennoscandia as the apex areas of the northward end-glacial colonization; and central (n = 8), including samples from the area between southern and northern regions, which has a mixed history of glacial survival and post-glacial colonization (Filipi et al, 2015; Marková et al, 2020)
The Bioanalyzer electropherogram for each sample showed a clear presence of two peaks expected for the two large rRNA species (18S and 28S), without any evidence of degraded products, and the average RNA Integrity Number was 8.92 ± 0.5
Summary
Regulatory variation in gene expression is increasingly being recognized as a major determinant of phenotypic variation (Lai et al, 2008; Fraser, 2013; Sandkam et al, 2015; Lin et al, 2017). The role of regulatory changes in the evolution of natural populations has not been firmly established, in part because the majority of gene expression studies have been conducted with model organisms in controlled laboratory settings (Nuzhdin et al, 2004; Fraser et al, 2010; Lin et al, 2017). Such studies explore the effect of genotype and physiological state of the organism on gene expression, but their generality is limited by the restricted range of genotypes and phenotypes available in laboratory populations (Lai et al, 2008). Compared to laboratory animals, there are potentially additional sources of variation in natural populations (Huang and Agrawal, 2016), due to genetic background differences or environmental heterogeneity (Harrison et al, 2012; Lenz, 2015), and careful selection of RGs is critical
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