Quantitative real-time RT-PCR (qRT-PCR)

Frozen colon tumor samples and matched controls were thawed, and mRNA was extracted using the RNeasy kit (Qiagen, Valencia, CA, USA). RNA (2 μg) was reverse-transcribed using SuperScript III (Life Technologies, Grand Island, NY, USA). EOMES, BMF and BIRC5 mRNA levels were measured by qRT-PCR and normalized to ACTB. Forty cycles of PCR (95 °C/10 s, 58 °C/10 s, 72 °C/10 s) were run on a LightCycler 480 II system (Roche, Indianapolis, USA), in 20 μl total reaction volume containing cDNA, SYBR Green I dye (Roche), and target-specific primers. The amount of specific mRNA was quantified by determining the point at which the fluorescence accumulation entered the exponential phase (C_t), and the C_t ratio of the target gene to ACTB was calculated for each sample. At least three separate experiments were performed for each sample.