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.