Real-time Quantitative PCR

RNA was isolated from PC3 cells with the ZR RNA MicroPrep RNA isolation kit (Zymo Research) according to the manufacturer’s protocol. For quantitative analysis of mRNA expression, comparative real-time PCR was performed with the use of Power SYBR Green RNA-to-CT 1 step kit (Applied Biosystems). Quantitative PCR was performed under the following conditions: 48 °C for 30 min, 95 °C for 10 min, then 40 cycles with 95 °C for 15 sec and 60°C for 1 min. Following the final cycle, the melt curves of the PCR products were determined to verify the integrity of the PCR products. The sequences for the amplification of human PAI-1 were: 5′-ACCGCAACGTGGTTTTCTCA-3′ (forward) and 5′-TTGAATCCCATAGCTGCTTGAAT-3′ (reverse). The sequences for the amplification of human TGFβ2 were: 5′-CAGCACACTCGATATGGACCA-3′ (forward) and 5′-CCTCGGGCTCAGGATAGTCT-3′ (reverse). The sequences for the amplification of human FGF1 were: 5′-ACACCGACGGGCTTTTATACG-3′ (forward) and 5′-CCCATTCTTCTTGAGGCCAAC-3′ (reverse). 18S rRNA was used as an external endogenous standard. The forward, 5′-CGGCTACATCCAAGGAA-3′, and reverse, 5′-GCTGGAATTACCGCGGCT-3′.