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Selection of suitable reference genes for normalization of genes of interest in canine soft tissue sarcomas using quantitative real‐time polymerase chain reaction
Author(s) -
Zornhagen K. W.,
Kristensen A. T.,
Hansen A. E.,
Oxboel J.,
Kjær A.
Publication year - 2015
Publication title -
veterinary and comparative oncology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.864
H-Index - 34
eISSN - 1476-5829
pISSN - 1476-5810
DOI - 10.1111/vco.12108
Subject(s) - reference genes , gene , polymerase chain reaction , normalization (sociology) , biology , real time polymerase chain reaction , primer (cosmetics) , reverse transcriptase , reverse transcription polymerase chain reaction , microbiology and biotechnology , gene expression , computational biology , genetics , chemistry , organic chemistry , sociology , anthropology
Quantitative real‐time reverse transcription polymerase chain reaction ( RT‐qPCR ) is a sensitive technique for quantifying gene expression. Stably expressed reference genes are necessary for normalization of RT‐qPCR data. Only a few articles have been published on reference genes in canine tumours. The objective of this study was to demonstrate how to identify suitable reference genes for normalization of genes of interest in canine soft tissue sarcomas using RT‐qPCR . Primer pairs for 17 potential reference genes were designed and tested in archival tumour biopsies from six dogs. The geNorm algorithm was used to analyse the most suitable reference genes. Eight potential reference genes were excluded from this final analysis because of their dissociation curves. β ‐Glucuronidase ( GUSB ) and proteasome subunit, beta type, 6 ( PSMB6 ) were most stably expressed with an M value of 0.154 and a CV of 0.053 describing their average stability. We suggest that choice of reference genes should be based on specific testing in every new experimental set‐up.