Abstract
BackgroundGene expression analysis is an important tool in contemporary research, with real-time PCR as the method of choice for quantifying transcription levels. Co-analysis of suitable reference genes is crucial for accurate expression normalisation. Reference gene expression may vary, e.g., among species or tissues; thus, candidate genes must be tested prior to use in expression studies. The domestic cat is an important study subject in both medical research and veterinary medicine. The aim of the present study was to develop TaqMan® real-time PCR assays for eight potential reference genes and to test their applicability for feline samples, including blood, lymphoid, endocrine, and gastrointestinal tissues from healthy cats, and neoplastic tissues from FeLV-infected cats.ResultsRNA extraction from tissues was optimised for minimal genomic DNA (gDNA) contamination without use of a DNase treatment. Real-time PCR assays were established and optimised for v-abl Abelson murine leukaemia viral oncogene homolog (ABL), β-actin (ACTB), β-2-microglobulin (B2M), β-glucuronidase (GUSB), hydroxymethyl-bilane synthase (HMBS), hypoxanthine phosphoribosyltransferase (HPRT), ribosomal protein S7 (RPS7), and tryptophan 5-monooxygenase activation protein, zeta polypeptide (YWHAZ). The presence of pseudogenes was confirmed for four of the eight investigated genes (ACTB, HPRT, RPS7, and YWHAZ). The assays were tested together with previously developed TaqMan® assays for feline glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and the universal 18S rRNA gene. Significant differences were found among the expression levels of the ten candidate reference genes, with a ~106-fold expression difference between the most abundant (18S rRNA) and the least abundant genes (ABL, GUSB, and HMBS). The expression stability determined by the geNorm and NormFinder programs differed significantly. Using the ANOVA-based NormFinder program, RPS7 was the most stable gene in the tissues studied, followed by ACTB and ABL; B2M, HPRT, and the 18S rRNA genes were the least stable ones.ConclusionThe reference gene expression stability varied considerably among the feline tissues investigated. No tested gene was optimal for normalisation in all tissues. For the majority of the tissues, two to three reference genes were necessary for accurate normalisation. The present study yields essential information on the correct choice of feline reference genes depending on the tissues analysed.
Highlights
Gene expression analysis is an important tool in contemporary research, with real-time PCR as the method of choice for quantifying transcription levels
Healthy cats were available from negative control groups, and they were sacrificed for reasons unrelated to this study
For the other four newly developed assays (ACTB, hypoxanthine phosphoribosyltransferase (HPRT), ribosomal protein S7 (RPS7), and tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein (YWHAZ)), we demonstrated the presence of processed pseudogenes
Summary
Gene expression analysis is an important tool in contemporary research, with real-time PCR as the method of choice for quantifying transcription levels. The domestic cat is an important study subject in veterinary medicine and in medical research It plays an essential role as a laboratory model for human infectious, hereditary and endocrine diseases and allows the study of topics such as host-pathogen interactions, defence mechanisms, and development of prophylactic or therapeutic regimens. Models under investigation include the glycogen storage disease type IV reported in the Norwegian Forest cat, the only reported animal model for this pathology [6], or the obesity-associated form of diabetes mellitus in the domestic cat that is similar to the type 2 diabetes mellitus in humans [7] For the latter, the domestic cat presents a valuable model for understanding the molecular mechanisms linking obesity to the development of insulin resistance, hypertension, and atherosclerosis [7]. The potential of the cat as an animal model and the similarities in genome organisation between humans and felids [8,9] provide the basis for a wide range of gene expression studies
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