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Reference gene validation for gene expression normalization in canine osteosarcoma: a geNorm algorithm approach


Selvarajah, Gayathri Thevi and Bonestroo, Floor A. S. and Timmermans Sprang, Elpetra P. M. and Kirpensteijn, Jolle and Mol, Jan A. (2017) Reference gene validation for gene expression normalization in canine osteosarcoma: a geNorm algorithm approach. BMC Veterinary Research, 13 (354). pp. 1-8. ISSN 1746-6148


BACKGROUND: Quantitative PCR (qPCR) is a common method for quantifying mRNA expression. Given the heterogeneity present in tumor tissues, it is crucial to normalize target mRNA expression data using appropriate reference genes that are stably expressed under a variety of pathological and experimental conditions. No studies have validated specific reference genes in canine osteosarcoma (OS). Previous gene expression studies involving canine OS have used one or two reference genes to normalize gene expression. This study aimed to validate a panel of reference genes commonly used for normalization of canine OS gene expression data using the geNorm algorithm. qPCR analysis of nine canine reference genes was performed on 40 snap-frozen primary OS tumors and seven cell lines. RESULTS: Tumors with a variety of clinical and pathological characteristics were selected. Gene expression stability and the optimal number of reference genes for gene expression normalization were calculated. RPS5 and HNRNPH were highly stable among OS cell lines, while RPS5 and RPS19 were the best combination for primary tumors. Pairwise variation analysis recommended four and two reference genes for optimal normalization of the expression data of canine OS tumors and cell lines, respectively. CONCLUSIONS: Appropriate combinations of reference genes are recommended to normalize mRNA levels in canine OS tumors and cell lines to facilitate standardized and reliable quantification of target gene expression, which is essential for investigating key genes involved in canine OS metastasis and for comparative biomarker discovery.

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Additional Metadata

Item Type: Article
Divisions: Faculty of Veterinary Medicine
DOI Number: https://doi.org/10.1186/s12917-017-1281-3
Publisher: BioMed Central
Keywords: Bone tumor; Dog; Osteosarcoma; Quantitative real-time PCR; Reference genes
Depositing User: Mas Norain Hashim
Date Deposited: 28 Sep 2018 10:37
Last Modified: 28 Sep 2018 10:37
Altmetrics: http://www.altmetric.com/details.php?domain=psasir.upm.edu.my&doi=10.1186/s12917-017-1281-3
URI: http://psasir.upm.edu.my/id/eprint/62931
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