Evaluation of critical design parameters for RT‐qPCR‐based analysis of multiple dUTPase isoform genes in mice

Abstract

The coupling of nucleotide biosynthesis and genome integrity plays an important role in ensuring faithful maintenance and transmission of genetic information. The enzyme dUTPase is a prime example of such coupling, as it generates dUMP for thymidylate biosynthesis and removes dUTP for synthesis of uracil‐free DNA. Despite its significant role, the expression patterns of dUTPase isoforms in animals have not yet been described. Here, we developed a detailed optimization procedure for RT‐qPCR‐based isoform‐specific analysis of dUTPase expression levels in various organs of adult mice. Primer design, optimal annealing temperature, and primer concentrations were specified for both nuclear and mitochondrial dUTPase isoforms, as well as two commonly used reference genes, GAPDH and PPIA. The linear range of the RNA concentration for the reverse transcription reaction was determined. The PCR efficiencies were calculated using serial dilutions of cDNA. Our data indicate that organs involved in lymphocyte production, as well as reproductive organs, are characterized by high levels of expression of the nuclear dUTPase isoform. On the other hand, we observed that expression of the mitochondrial dUTPase isoform is considerably increased in heart, kidney, and ovary. Despite the differences in expression levels among the various organs, we also found that the mitochondrial dUTPase isoform shows a much more uniform expression pattern as compared to the reference genes GAPDH and PPIA.

Genomic uracil occurence and the role of dUTPase in transgenic models

“…However, in other mouse tissues, no signal (in case of lung) or only weak band (in case of heart) could be found hampering correct comparison of WT and D47 strains. These results are in good agreement with the recent finding that expression pattern of dUTPase is highly organ-specific 114 . Further details of gene expression may be explored using quantitative real-time PCR (qPCR) and proper statistical analysis.…”

Redox status of cysteines does not alter functional properties of human dUTPase but the Y54C mutation involved in monogenic diabetes decreases protein stability

“…The dUTPase enzymes play a dual role in sanitizing the nucleotide pool and in providing precursor for thymidylate biosynthesis 11 , 12 . It has been established that in humans and in several other eukaryotes, two dUTPase isoforms are present, one nuclear and one mitochondrial and their expression is under control of alternative promoters 13 , 14 . The human dUTPase isoforms differ only in their respective intracellular trafficking signals: a well characterized nuclear localization signal 13 , 15 for the nuclear isoform and a mitochondrial leader sequence for the mitochondrial isoform 13 (Fig.…”

Discovery of two new isoforms of the human DUT gene

“…In contrast to its indispensable nature, the isoform distribution of dUTPase was investigated in only a few model organisms. In mouse, one nuclear and one mitochondrial isoform of dUTPase were described—similar to humans 6 , 7 , 27 . In Drosophila melanogaster, one nuclear and one cytoplasmic dUTPase isoform was shown to exist 48 , 49 .…”

“…To verify the specificity of the PCR products regarding the four isoforms, we sent PCR products for Sanger sequencing, however as the length of all PCR products are below 90 base pairs, a nested PCR design was applied 27 . A longer PCR product was generated for each target with another reverse primer and the appropriate forward primer and these products were purified and analysed with Sanger sequencing.…”