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FEBS Open Bio. 2019 Jun;9(6):1153-1170. doi: 10.1002/2211-5463.12654. Epub 2019 May 29.

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

Author information

1
Institute of Enzymology, RCNS, Hungarian Academy of Sciences, Budapest, Hungary.
2
Department of Applied Biotechnology and Food Sciences, Budapest University of Technology and Economics, Budapest, Hungary.
3
Department of Animal Biotechnology, Agricultural Biotechnology Institute, National Agricultural Research and Innovation Centre, Gödöllő, Hungary.
4
Faculty of Agricultural and Environmental Science, Szent István University, Gödöllő, Hungary.

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.

KEYWORDS:

RT-qPCR optimization; dUTPase; isoform-specific expression levels; mitochondrial dUTPase isoform; mouse organs; nuclear dUTPase isoform

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