RNA isolation and Real-time PCR
Cells were collected for RNA extraction, and supernatants were collected
to detect the cytokine levels. Total RNA was isolated using TRIzol
Reagent (Invitrogen, Carlsbad, CA, USA), and reverse transcription was
performed using the cDNA synthesis kit according to the manufacturer’s
instructions (Takara, Dalian, China). Real-time PCR was conducted using
BRYT Green. The primers used were as follows: β-actin F:
5’-GGATGCAGAAGGAGATCACTG-3’, R:
5’-CGATCCACACGGAGTACTT-3’; IL2RA F:
5’-ATGGCTGCAACCATGGAGAC-3’, R: 5’-TCTGTTCCCGGCT TCTTACC-3’. Real-time
PCR parameters were as follows: step one, one cycle at 95°C for 10 min;
step two, 40 cycles at 95°C for 15 s, 59°C for 1 min; step three, 95°C
for 15 s, 60°C for 15 s and 95°C for 15 s. All of the reactions of
real-time PCR were performed in triplicate. The relative expression of
candidate genes was calculated using the 2-ΔΔCtmethod.