RNA isolation and qRT-PCR
Identic to the manufacturer’s procedure, whole cellular RNA (1 µg) was extracted from cells using TRIzol reagent, then assessed the quantity and quality of RNA samples on Nanodrop and gel electrophoresis. Only RNA samples with RNA Integrity Numbers (RIN) > 6 were included in the analyses. Then reverse transcriptional reaction was conducted to obtain cDNA by Prime Script RT Master Mix. According to the manufacturer’s instructions, cDNA was synthesized from 1 μg of total RNA with an RT-for-PCR kit (Takara Bio, Inc., Otsu, Japan). Primers (PRF1, PIK3B, PD-1, AKT-1, FAS-L, TRAIL, GERA & B) were designed and certified using NCBI-Primer BLAST. Specimens were duplicated from three independent trials; β2-microglobulin RNA levels were employed as an internal reference for all experiments. The relative expression levels of genes were calculated using the 2−ΔΔCT methods.