4.4. cDNA Synthesis and Quantitative Polymerase Chain Reaction

The expression levels of immune response-related genes, including T-cell-related clusters of differentiation antigens and biomarkers, were measured by quantitative polymerase chain reaction (qPCR) using the Bio-Rad CFX96 system (Bio-Rad, Hercules, CA, USA). PCR primers for CD8B and GAPDH were purchased from Takara Bio Inc. (Shiga, Japan), and the primers for PD-L1 were obtained from a previous report [40]. The sequences of the primers were as follows:

CD8B-forward 5′-GGCATCTACTTCTGCATGATCGTC-3′

CD8B-reverse 5′-TGGGTAACCGGCACACTCTC-3′

PD-L1-forward 5′-AAATGGAACCTGGCGAAAGC-3′

PD-L1-reverse 5′-GATGAGCCCCTCAGGCATTT-3′

GAPDH-forward 5′-GCACCGTCAAGGCTGAGAAC-3′

GAPDH-reverse 5′-ATGGTGGTGAAGACGCCAGT-3′.

cDNAs were synthesized from 500 ng of total RNA isolated from each specimen of ESCC or OSCC using the Prime Script RT reagent Kit (Takara, Shiga, Japan) for each of the 10 qPCR reactions. The PCR mixture consisted of 5 μL of SsoFast EvaGreen Supermix (Bio-Rad, Hercules, CA, USA), 3.5 μL of RNase/ DNase-free water, 0.5 μL of 5-μM primer mix, and 1-μL cDNA in a final total volume of 10 μL. This was then applied to the CFX96 system. Cycling conditions were as follows: 30 s at 95 °C, followed by 50 cycles of 1 s at 95 °C and 5 s at 60 °C. At the end of each program, a melting curve analysis was performed from 70 °C to 94 °C to confirm homogeneity of the PCR products. All assays were performed in triplicate, and mean values were used to calculate the gene expression levels. For calibration of the absolute quantification, a standard curve for each target was prepared by five 10-fold serial dilutions of the standard nucleotide that was generated by the linearized plasmid cloned with the PCR product of each target. This was confirmed by DNA sequencing using the Sanger method and was quantified by SmartSpec3000 (Bio-Rad, Hercules, CA, USA).