Quantitative PCR

A total of 2.0 × 10⁴ cells were seeded onto a single collagen I-coated well of a 96-well plate, and simultaneously transfected with siRNAs (targeting AMAP1 or PIAS3, or a negative control). Cell lysates were prepared 72 h after seeding, and reverse transcription was performed using Cell Amp™ Direct Probe RT-qPCR Kit (Takara). Quantitative PCR (qPCR) was performed with the obtained samples following the manufacturer's instructions. The following fluorescent qPCR probes were used: CA9 TaqMan® Gene Expression Assays, FAM-MGB (Hs00154208_m1); HIF1A TaqMan® Gene Expression Assays, FAM-MGB (Hs00153153_m1); ACTB TaqMan® Gene Expression Assays, VIC-MGB_PL (Hs99999903_m1). One of the FAM probes (for either CA9 or HIF1A) was duplexed with the VIC probe (for ACTB), and the expression of CA9 and HIF1A was normalized using that of ACTB. Assays were performed using LightCycler® 96 System (Roche).