Quantitect reverse transcription kit

Trizol (Invitrogen, Carlsbad, CA, USA) was used to extract total RNA from chicken DF-1 cells. Total RNA was quantified by NanoDrop spectrophotometer (Thermo Fisher Scientific Inc., Waltham, MA, USA). After quantification, 2 μg of total RNA was reverse-transcribed to synthesize cDNA using the using QuantiTect Reverse Transcription Kit (Toyobo, Osaka, Japan) according to the manufacturer’s instructions.

Quantitative real-time PCR was performed as described previously^{14 (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.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′