Zornhagen K W, Kristensen A T, Hansen A E, Oxboel J, Kjaer A
Department of Veterinary Clinical and Animal Sciences, Faculty of Health and Medical Sciences, University of Copenhagen, Frederiksberg C, Denmark.
Department of Clinical Physiology, Nuclear Medicine & PET, Copenhagen University Hospital and Cluster for Molecular Imaging, Faculty of Health Sciences, University of Copenhagen, Copenhagen N, Denmark.
Vet Comp Oncol. 2015 Dec;13(4):485-93. doi: 10.1111/vco.12108. Epub 2014 Jul 4.
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.
定量实时逆转录聚合酶链反应（RT-qPCR）是一种用于定量基因表达的灵敏技术。稳定表达的内参基因对于RT-qPCR数据的标准化是必要的。关于犬类肿瘤内参基因的文章仅有少数发表。本研究的目的是展示如何使用RT-qPCR鉴定适合用于犬软组织肉瘤中感兴趣基因标准化的内参基因。设计了针对17个潜在内参基因的引物对，并在来自6只犬的存档肿瘤活检样本中进行了测试。使用geNorm算法分析最合适的内参基因。由于解离曲线的原因，8个潜在内参基因被排除在最终分析之外。β-葡萄糖醛酸酶（GUSB）和蛋白酶体亚基β型6（PSMB6）表达最稳定，其M值为0.154，CV为0.053，描述了它们的平均稳定性。我们建议内参基因的选择应基于每个新实验设置中的具体测试。
