SSC was removed, and 75 µL reverse transcription Master Mix (Sigma-Aldrich) was added to each well. Reverse transcription was conducted according to the manufacturer’s protocol. After reverse transcription, the wells were washed with 0.1× SSC. Sections were then incubated in 75 µL 0.08 M KOH for 5 min at room temperature, and then were incubated in 75 µL Second Strand Mix (Thermo Fisher Scientific, Waltham, MA, USA) for 15 min at 65 °C. After removal of the Second Strand Mix, 100 µL Buffer EB were added, and sections were placed in 35 µL 0.08 M KOH for 10 min at room temperature. The samples were then transferred from each well to a corresponding tube containing Tris-HCl (1 M, pH 7.0). Next, 1 µL of the sample was added to the qPCR plate well containing the KAPA SYBR FAST qPCR Master Mix (KAPA Biosystems, Cape Town, South Africa). The qPCR was performed following the manufacturer’s protocol, and the optimal number of cycles was determined. Next, 65 µL of the cDNA Amplification Mix (Takara Bio, Mountain View, CA, USA) was added to the remaining sample, which was then incubated for 12 cycles according to the recommended protocol.

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