2.9∣RNA isolation and real-time quantitative PCR
Total RNA was extracted from splenocytes of individual mice after
stimulation with 1 μg/mL of rFor t 2 or medium alone for 3 days using
TRIzol reagents. First-strand cDNA synthesis was carried out using
SuperScript III kit (Invitrogen, Carlsbad, CA). The analyzed target
genes were IL-10, IL-13, IFN-γ, and FOXP3, and these specific primers
are given in supplementary Table 1. PCR reactions were run on an ABI
StepOnePlus machine (Life Technologies) as follows: an initial 10-minute
step at 95℃ followed by 40 cycles of 95℃ for 15 seconds and 60℃ for 1
minute. The fluorescent signal from SYBR Green was detected immediately
after the extension step of each cycle, and the cycle at which the
product was first detectable was recorded as the cycle threshold. Data
were imported into an Excel database and analyzed using the comparative
cycle threshold method with normalization of the raw data to β-actin.