Ab 7500 fast qpcr system

The expression levels of microtubule-associated protein 1A/1B-light chain (LC3B) and beclin-1 (BECN-1) were assessed using a real-time polymerase chain reaction (qPCR) with the SensiFAST SYBR Hi-ROX kit (Bioline USA, Taunton, MA, USA) and Applied Biosystems (AB) 7500 Fast qPCR System (Applied Biosystems, Foster City, CA, USA). This qPCR was performed four times. The target gene expression was normalized to the expression of the reference housekeeping gene GAPDH. The normalized cycle threshold of the normal sham group was used to calculate the fold difference for each group. We used several primers as follows; LC3B forward: 5′-CGGAGCTTCGAACAAAGAGT-3′, LC3B reverse: 5′-TCTCACCCTTGTATCGCTCT-3′, BECN-1 forward: 5′-TTCAAGACTTCGGACCGCGT-3′, BECN-1 reverse: 5′-AGTTAGCCTCTTCCTCCTGG-3′, GAPDH forward: 5′-CTGAGAATGGGAAGCTGGTC-3′, GAPDH reverse: 5′-CTCCACGACATACTCAGCAC-3′.

IL-10 and IL-18 mRNA expression levels were assessed by qPCR using a SensiFAST SYBR Hi-ROX kit (Bioline USA, Taunton, MA, USA) and AB7500 Fast qPCR System (Applied Biosystems, Foster City, CA, USA). Seven tumor tissues per group were analyzed in quadruplicate. Target genes were normalized to the reference housekeeping gene GAPDH. Fold differences were calculated for each group using the normalized CT values for the normal-sham group. The primers used are listed in Supplementary Table S1.