mRNA from canine tumors and brain tissues was isolated using an RNeasy kit (Qiagen) according to the manufacturer’s instructions, and all mRNA samples were stored at −80°C before analyses. An iTaq Universal one-step kit (Bio-Rad) was used to perform the RT-qPCR, with β-actin serving as an internal control. The primers for uPAR (NCBI-ID XM_014119899.1) were 5′-GCCTTACCGAGGTTGTGTGT-3′ (forward) and 5′-CATCCAGGCACTGTTCTTCA-3′ (reverse). The β-actin-specific primers were 5′-CTGGAACGGTGAAGGTGACA-3′ (forward) and 5′-GGGAGAGGACTGGGCCATT-3′ (reverse). The uPAR primers used in this study were created using the Primer3Plus platform.17 (link) The specificity of the uPAR primers and their corresponding amplicons were evaluated in silico using the BLAST online tool.18 (link) uPAR mRNA expression was calculated as the fold-change relative to the β-actin control using comparative CT methodology.9 (link)
Protocol detail
Quantifying uPAR mRNA Expression in Canine Tumors
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Other organizations : Atrium Health Wake Forest Baptist, Virginia–Maryland College of Veterinary Medicine, Virginia Tech, Virginia Tech - Wake Forest University School of Biomedical Engineering & Sciences
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Variable analysis
independent variables
- None explicitly mentioned
- UPAR mRNA expression
- β-actin as internal control
- Positive control: none mentioned
- Negative control: none mentioned
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