Sybr green premix pcr master mix

Total RNA of Nyth-ori-3-1, TPC-1, and BCPAP cells was extracted utilizing Trizol method and 500 ng total RNA was reversely transcribed into cDNA with “PrimeScript^TM RT reagent Kit with gDNA Eraser” (Takara, Japan). Quantitative real time-PCR (qRT-PCR) was perform using “SYBR Green Premix PCR Master Mix” (Takara, Japan) according to the manufacturer protocols. We calculated the relative mRNA expression markers utilizing the Ct method (2^−ΔΔCt) after being normalized to β-actin. All reactions were carried out independently and repeated three times each time. A primer sequence of the six genes was used and is presented in the Supplementary Table 2.

Total RNA was extracted with TRIzol reagent and then reversely transcribed into cDNA using a Pria Revert Aid First Strand cDNA Synthesis Kit (catalog number: #A5001, promega). Following the manufacturer’s protocols, Real-time PCR was performed using a SYBR Green Premix PCR Master Mix (catalog number: #DRR041B, TAKARA). Relative mRNA expression of CALM1 and EGFR was calculated using the 2^−ΔΔCt method after being normalized to GAPDH, which served as internal loading control. PCR was performed with the following primer sets: CALM1 forward, 5′-GGTCAGAACCCAACAGAA-3′ and reverse, 5′-AGACTCGGAATGCCTCA-3′; and EGFR forward, 5′-AGGCACGAGTAACAAGCTCAC-3′ and reverse, 5′-ATGAGGACATAACCAGCCACC-3′. GAPDH forward, 5′-TGACTTCAACAGCGACACCCA-3′ and reverse, 5′-CACCCTGTTGCTGTAGCCAAA-3′. All experiments were performed independently three times and shown was the representative one.