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). However, 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). 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). Our results underscore the need of a systematic evaluation of appropriate RGs to each particular experimental system and provide a useful starting point for further studies. In contrast, the traditional housekeeping genes Actb, Gapdh, Sdha, and Tuba1a displayed large expression variation and are not recommended as internal controls. The combinations Ube2d2a and Ppia or Ube2d2a and Tbp were identified to be sufficient for the normalization. Ube2d2a, Ppia, and Tbp were consistently identified as the least variable genes, followed by Rn18s, Ywhaz, and Rplp0. 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. Thus, rigorous determination of RGs in natural populations of the bank vole was necessary to facilitate gene expression studies in this emerging model species. 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. 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. 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. 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. 2Laboratory of Molecular Ecology, Institute of Animal Physiology and Genetics of the Czech Academy of Sciences, Liběchov, Czechia.1Laboratory of Developmental Biology, Institute of Animal Physiology and Genetics of the Czech Academy of Sciences, Liběchov, Czechia.Without Q-Bond, the primer and polymerase bind sequentially to the template, increasing primer annealing time.Lucie Němcová 1† Silvia Marková 2† Petr Kotlík 2* In addition, the unique buffer composition supports the melting of DNA, reducing denaturation and extension times. The amplification plots for the duplex reactions (colored) overlapped with those for control singleplex reactions (gray), demonstrating the reliability of the duplex analysis.| Q-Bond in Rotor-Gene Multiplex PCR Master Mix increases the affinity of DNA polymerase for short single-stranded DNA, reducing primer annealing time to a few seconds. Triplicate reactions were run on the Rotor-Gene 3000 using leukocyte RNA as template (10-fold dilutions from 100 ng to 10 pg). Commercial Partner and Distributor Solutionsĭuplex, real-time one-step RT-PCR was performed using the Rotor-Gene Multiplex RT-PCR Kit and self-designed TaqMan assays for 28S rRNA (Cyanine 670 dye data in inset) and PPIA (cyclophilin A, FAM dye).Quality Assurance and Environmental, Health & Safety.Quality, Environmental, Health & Safety.Solutions for Laboratory-Developed Tests. Whole Genome/Transcriptome Amplification.SYBR Green- or Dye-Based One-Step qRT-PCR.Reverse Transcription & cDNA Synthesis for qPCR.Enzymes cloning and recombinant DNA technology.Enzymes reaction cleanup and yield improvement.Tagged Protein Expression, Purification, Detection.
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