Total RNA from normal esophageal samples (n = 10) and ESCC samples (n = 10) were isolated using RNeasy Mini Kit (Cat.74101, Qiagen, Germany) according to the manufacturer’s instruction. The synthesis of cDNA used for genes were finished using the BestarTM qPCR RT kit (DBI; #DBI-0) from 2µg RNA. The relative mRNA levels of SPP1, MMP12, COL10A1 and COL5A2 were determined by qRT-PCR method using a 20µL reaction system. The PCR process was done on an ABI PRISM 7500 real-time PCR system (Applied Biosystems, Carlsbad, CA, USA) using the following settings: 95℃ for 2 min, followed by 40 cycle of 94℃ for 20 S, 58℃ for 20 S and 72℃ for 20s. GAPDH was used as the internal normalized reference to genes. The fold change was determined via 2 − ΔΔCt (ΔΔCt = (ΔCt of genes of interest) − (ΔCt of GAPDH). The primer sequences used as follows: SPP1: F: 5’-TTTGTTGTAAAGCTGCTTTTCCTC-3’R: 5’-GAATTGCAGTGATTTGCTTTTGC-3’; MMP12: F: 5’-ACGTGGCATTCAGTCCCTGT-3’R: 5’-AACACTGGTCTTTGGTCTCTCAGAA-3’; COL10A1: F: 5’-ATGCTGCCACAAATACCCTTT-3’R: 5’-GGTAGTGGGCCTTTTATGCCT-3’; COL5A2: F: 5’-GGAAGAAGACGAGGATGAAGGATA-3’; R: 5’-CAGGAC CAGAAGGACCAACT-3’.

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