Rneasy plus mini kit 50 kit

The RNeasy Plus Mini Kit (50) kit (Qiagen, Hilden, Germany) was used to extract total RNA, which was then reverse transcribed with the Advantage RT-for-PCR Kit (Qiagen) in accordance with the manufacturer's protocols. The target sequence was amplified with real-time PCR with the SYBR Green PCR Kit (Qiagen). The cycling parameters used were 95 °C for 15 s, 55-60 °C for 15 s, and 72 °C for 15 s for 45 cycles. Melting curve analyses were performed, and Ct values were determined during the exponential amplification phase of real-time PCR. SDS 1.9.1 software (Applied Biosystems, Massachusetts, USA) was used to evaluate amplification plots. The 2^-ΔΔCt method was used to determine relative fold changes in target gene expression in cell lines, which was normalized to expression levels in corresponding control cells (defined as 1.0). The equation used was 2^-ΔΔCt (ΔCt = ΔCt target - ΔCt GAPDH; ΔΔCt = ΔCt expressing vector - ΔCt control vector). When calculating relative expression levels in surgically extracted CRC samples, relative fold changes in target gene expression were normalized to expression values in normal colon epithelial tissues (defined as 1.0) using the following equation: 2 ^-ΔΔCt (ΔΔCt = ΔCt tumor - ΔCt nontumor). All experiments were performed in duplicate. Table S5 lists all sequences of all primers used.