Quantitative real-time PCR was performed as described previously14 (link). Total RNA was extracted from 2 × 105 of 2.5D organoid cells at early and late passage, 3D organoids, and urothelial carcinoma cell lines by using a NucleoSpin kit (Takara Bio Inc., Shiga, Japan) following the manufacturer’s protocol. First-strand cDNA was prepared using a QuantiTect Reverse Transcription Kit (TOYOBO, Tokyo, Japan). Quantitative real-time PCR was done using a QuantiTect SYBR I Kit (QIAGEN, Hilden, Germany) and a StepOnePlus Real-Time PCR System (Applied Biosystems, Waltham, MA, USA). The ΔΔCq method was used for quantification. Specific primers used for dog BC stem cell markers, SOX2, CD44, and GAPDH were designed and are shown in Table 3 .
Primers for real-time quantitative PCR analysis.
| Gene name | Primer | Sequence |
|---|---|---|
| SOX2 | Forward | 5′-GCCCTGCAGTACAACTCCAT-3′ |
| Reverse | 5′-GGAGTGGGAGGAGGAGGTAA-3′ | |
| CD44 | Forward | 5′-CCAAGACAGTTCCAGGGTGT-3′ |
| Reverse | 5′-TTGAGGTTTCCGCATAGGAC-3′ | |
| GAPDH | Forward | 5′-AACTCCCTCAAGATTGTCAGCAA-3′ |
| Reverse | 5′-CATGGATGACTTTGGCTAGAGGA-3′ |
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