Hindawi Publishing Corporation Molecular Biology International Volume 2013, Article ID 587680, 7 pages http://dx.doi.org/10.1155/2013/587680 Research Article GAPDH Pseudogenes and the Quantification of Feline Genomic DNA Equivalents A. Katrin Helfer-Hungerbuehler, Stefan Widmer, and Regina Hofmann-Lehmann Clinical Laboratory, Vetsuisse Faculty, University of Zurich, Winterthurerstrasse 260, 8057 Zurich, Switzerland Correspondence should be addressed to A. Katrin Helfer-Hungerbuehler; khungerbuehler@vetclinics.uzh.ch Received 17 January 2013; Revised 21 March 2013; Accepted 28 March 2013 Academic Editor: Emanuel Strehler Copyright © 2013 A. Katrin Helfer-Hungerbuehler et al. Tis 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. 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 defned 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). Te 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 quantifcation 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 identifed, 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. 1. Introduction Fluorescence-based quantitative real-time PCR (qPCR) is a highly sensitive method for the detection and quantifcation of nucleic acids. Due to its conceptual simplicity, sensitivity, specifcity, and speed, qPCR applications can be found in a variety of felds, including medicine and the life sciences [1, 2]. In clinical diagnostics, qPCR is broadly used for the detection and quantifcation of bacterial and viral loads, gene dosage determination, cancer diagnostics, and applications in forensic medicine [3–7]. To assess the cell number present in a PCR reaction, the coanalysis of suitable reference genes is crucial. Such refer- ence genes should be single-copy number genes and should not frequently undergo genetic alterations, to allow the accurate normalization of genomic DNA (gDNA) samples. In addition, internal control genes are also used to investigate abnormalities in gene number, and amplifed oncogenes have been shown to have diagnostic, prognostic, and therapeutic relevance. Tus, TaqMan PCR-based gene quantifcation assays are also used to identify allelic imbalances (germ-line deletions or amplifcations), for example, in individuals suf- fering from breast cancer, cutaneous melanoma, or nervous system tumors [8, 9]. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) represents a universally expressed reference gene that has many biological roles in addition to its function in glycol- ysis. A GAPDH assay has been developed for the accurate normalization of feline messenger RNA (mRNA) expression [10], and this assay was recently validated and compared to other potential feline mRNA reference gene assays [11]. Subsequently, the GAPDH assay was also applied as quality control to test for the integrity of the gDNA and the absence of PCR inhibitors [10, 12, 13]. One report suggested that a single copy of the GAPDH pseudogene is present in the feline genome and that the feline GAPDH assay can therefore be used to quantify cell number in feline samples [14]. However, no information on the exact position or the sequence of this GAPDH pseudogene was provided [14]. In contrast, a variable number of GAPDH pseudogenes has been reported for other organisms [15, 16].