GAPDH Pseudogenes and the Quantification of Feline Genomic DNA EquivalentsReport as inadecuate




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Molecular Biology InternationalVolume 2013 2013, Article ID 587680, 7 pages

Research ArticleClinical Laboratory, Vetsuisse Faculty, University of Zurich, Winterthurerstrasse 260, 8057 Zurich, Switzerland

Received 17 January 2013; Revised 21 March 2013; Accepted 28 March 2013

Academic Editor: Emanuel Strehler

Copyright © 2013 A. Katrin Helfer-Hungerbuehler et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Quantitative real-time PCR qPCR is broadly used to detect and quantify nucleic acid targets. In order to determine cell copy number and genome equivalents, a suitable reference gene that is present in a defined number in the genome is needed, preferably as a single copy gene. For most organisms, a variable number of glyceraldehyde-3-phosphate dehydrogenase GAPDH pseudogenes have been reported. However, it has been suggested that a single-copy of the GAPDH pseudogene is present in the feline genome and that a GAPDH assay can therefore be used to quantify feline genomic DNA gDNA. The aim of this study was to determine whether one or more GAPDH pseudogenes are present in the feline genome and to provide a suitable alternative qPCR system for the quantification of feline cell copy number and genome equivalents. Bioinformatics and sequencing results revealed that not just one but several closely related GAPDH-like sequences were present in the cat genome. We thus identified, developed, optimized, and validated an alternative reference gene assay using feline albumin fALB. Our data emphasize the need for an alternative reference gene, apart from the GAPDH pseudogene, for the normalization of gDNA levels. We recommend using the fALB qPCR assay for future studies.





Author: A. Katrin Helfer-Hungerbuehler, Stefan Widmer, and Regina Hofmann-Lehmann

Source: https://www.hindawi.com/



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