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Quantitative TaqMan® real-time PCR assays for gene expression normalisation in feline tissues


Kessler, Y. Quantitative TaqMan® real-time PCR assays for gene expression normalisation in feline tissues. 2009, University of Zurich, Vetsuisse Faculty.

Abstract

Gene expression analysis is an important tool in contemporary research, with real-time PCR as the method of choice for accurate and fast quantification of mRNA transcription levels. Co-analysis of reference genes is crucial for expression normalization. Since, reference gene expression may vary, e.g., among different species and tissues, their applicability must be tested prior to use in expression studies. The domestic cat is an important study subject in medical research (e.g. animal model for infectious diseases or endocrine disorders) and in veterinary medicine. Little is known about feline reference genes and their application in TaqMan® real-time PCR assays. The aim of the present study was to develop TaqMan® assays for eight potential feline reference genes and to test their applicability for feline samples, including blood, lymphoid, endocrine, and gastrointestinal tissues from clinically healthy cats, and neoplastic tissues. RNA extraction from tissues was optimized for minimal gDNA contamination without use of a DNase treatment. Candidate reference genes included: ABL (v-abl Abelson murine leukemia viral oncogene homolog), ACTB (ß-actin), B2M (ß-2-microglobulin), GUSB (ß-glucuronidase), HMBS (hydroxymethyl-bilane synthase), HPRT (hypoxanthine phosphoribosyltransferase), RPS7 (ribosomal protein S7), YWHAZ (tryptophan 5-monooxygenase activation protein, zeta polypeptide). The assays were tested together with previously developed TaqMan® assays for feline GAPDH (glyceraldehyde-3-phosphate dehydrogenase) and the universal 18S rRNA gene. The suitability of the candidate genes was assessed using the geNorm and NormFinder programs. A significant difference was found among the expression levels of the ten candidate reference genes (pKW < 0.001): the expression levels for the 18S rRNA gene were > 106-times higher than those of ABL and HMBS. This will allow matching the expression level of the reference genes with that of the target genes. The presence of pseudogenes was confirmed for four of the eight tested genes. The study confirmed that reference gene expression stability varies considerably among the tested feline tissues. No reference gene was suitable for optimal gene expression normalization in all tissues. For the majority of the tissues, two to four reference genes were found to be a recommendable number of genes for accurate normalization. ACTB, RPS7, and ABL were among the most stable reference genes in the studied tissues, while HPRT, 18S rRNA gene and GAPDH were among the least stable ones. The present study yields essential information on the correct choice of feline reference genes depending on the tissues analyzed.

Abstract

Gene expression analysis is an important tool in contemporary research, with real-time PCR as the method of choice for accurate and fast quantification of mRNA transcription levels. Co-analysis of reference genes is crucial for expression normalization. Since, reference gene expression may vary, e.g., among different species and tissues, their applicability must be tested prior to use in expression studies. The domestic cat is an important study subject in medical research (e.g. animal model for infectious diseases or endocrine disorders) and in veterinary medicine. Little is known about feline reference genes and their application in TaqMan® real-time PCR assays. The aim of the present study was to develop TaqMan® assays for eight potential feline reference genes and to test their applicability for feline samples, including blood, lymphoid, endocrine, and gastrointestinal tissues from clinically healthy cats, and neoplastic tissues. RNA extraction from tissues was optimized for minimal gDNA contamination without use of a DNase treatment. Candidate reference genes included: ABL (v-abl Abelson murine leukemia viral oncogene homolog), ACTB (ß-actin), B2M (ß-2-microglobulin), GUSB (ß-glucuronidase), HMBS (hydroxymethyl-bilane synthase), HPRT (hypoxanthine phosphoribosyltransferase), RPS7 (ribosomal protein S7), YWHAZ (tryptophan 5-monooxygenase activation protein, zeta polypeptide). The assays were tested together with previously developed TaqMan® assays for feline GAPDH (glyceraldehyde-3-phosphate dehydrogenase) and the universal 18S rRNA gene. The suitability of the candidate genes was assessed using the geNorm and NormFinder programs. A significant difference was found among the expression levels of the ten candidate reference genes (pKW < 0.001): the expression levels for the 18S rRNA gene were > 106-times higher than those of ABL and HMBS. This will allow matching the expression level of the reference genes with that of the target genes. The presence of pseudogenes was confirmed for four of the eight tested genes. The study confirmed that reference gene expression stability varies considerably among the tested feline tissues. No reference gene was suitable for optimal gene expression normalization in all tissues. For the majority of the tissues, two to four reference genes were found to be a recommendable number of genes for accurate normalization. ACTB, RPS7, and ABL were among the most stable reference genes in the studied tissues, while HPRT, 18S rRNA gene and GAPDH were among the least stable ones. The present study yields essential information on the correct choice of feline reference genes depending on the tissues analyzed.

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Additional indexing

Item Type:Dissertation
Referees:Hofmann-Lehmann R, Ackermann M
Communities & Collections:05 Vetsuisse Faculty > Veterinary Clinic > Department of Farm Animals
Dewey Decimal Classification:570 Life sciences; biology
630 Agriculture
Date:2009
Deposited On:24 Feb 2010 13:57
Last Modified:07 Dec 2017 01:32
Number of Pages:66

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