Applied biosystems 7300 detection system

Total RNA was isolated using the TRIzol^® Reagent (Invitrogen), according to the manufacturer's recommended protocol, and 1 μg of RNA was reversely transcribed using the SuperScript First-Strand Synthesis System (Invitrogen, CA, USA). The RNA templates were treated with DNase I to avoid genomic DNA contamination. Real-time PCR analyses were performed using an Applied Biosystems 7300 Detection System (Applied Biosystems, CA, USA) using primers for HER3 (forward: 5′-CCTGGGACTCTGAATGGC-3′, reverse: 5′-AGCCTGTCACTTCTCGAATC-3′), HER2 (forward: 5′-AGCCCTGGTCACCTACAACA-3′, reverse: 5′-GCA CTGGTAACTGCCCTCAC-3′), GAPDH (forward: 5′-TG ACTTCAACAGCGACACCCA-3′, reverse: 5′-CACCC TGTTGCTGTAGCCAAA-3′). Real-time PCR was performed using the SYBR^@ Premix Ex Taq^TM kit according to the manufacturer's instructions (Takara, Japan). The data were normalized to the GAPDH levels in the samples and calculated using the ΔΔCt method.

Total RNA was isolated using Trizol reagent according to the manufacturer's protocol (Invitrogen, Carlsbad, CA). Reverse transcription reactions were performed using the AMV First Strand cDNA Synthesis Kit (NEB, USA). Real-time PCR assays were performed using an Applied Biosystems 7300 Detection System (Applied Biosystems®, CA). The real-time PCR reaction was performed according to the protocol of the Power SYBR Green PCR Master Mix (Applied Biosystems, CA). The amplification procedure: 95°C for 15 seconds, and 60°C for 60 seconds for 40 cycles. Data was analyzed using the Sequence Detection Software, Version 1.2.3 (Applied Biosystems). The amount of target cDNA was calculated and normalized with the GAPDH levels in the samples. The primers utilized for real-time PCR analysis were listed in Table S2.

Human tissue samples and all cells were homogenized in TRIzol (Invitrogen,Grand Island, NY, USA), and total RNA was isolated according to the manufacturer's suggested protocol: 1 μg of RNA was used for cDNA synthesis. To determine the relative level of cDNA, real-time PCR analyses were performed using an Applied Biosystems 7300 Detection System (Applied Biosystems®, CA). The real-time PCR reaction was performed according to the protocol of the SYBR® Premix Ex Taq™ kit (Takara, DRR041). Duplicate runs of each sample were normalized to β-actin to determine relative expression levels. All Primers were described in Table S4.