Total RNA was isolated from cells using Trizol reagent (Molecular Research Center
Inc., Cincinnati, OH, USA). RNA concentrations were measured from the absorbance
at 260 and 280 nm. Complementary DNA (cDNA) synthesis was performed using a
Maxime RT PreMix Kit (Intron Biotechnology, Seoul, South Korea), in accordance
with the manufacturer’s protocol. The following primers were used for real-time
RT-PCR analyses: IL-6, 5′-GTA CTC CAG AAG ACC AGA GG-3′ and 5′-TGC TGG TGA CAA
CCA CGG CC-3′; IL-1β, 5′-CAG GAT GAC ATG AGC ACC-3′ and 5′-CTC TGC AGA CTC AAA
CTC CAC-3′; TNF-α, 5′-TTG ACC TCA GCG CTG AGT TG-3′ and 5′-CCT GTA GCC CAC GTC
GTA GC-3′; and β-actin, 5′-AGG CTG TGC TGT CCC TGT AT-3′ and 5′-ACC CAA GAA GGA
AGG CTG GA-3′. The levels of IL-1β, IL-6, TNF-α, and β-actin mRNAs were measured
using a Chromo 4TM Real-Time PCR Detector (Bio-Rad Laboratories Inc., Hercules,
CA, USA). For relative quantification, the reactions were performed using 2×
iQTM SYBR Green Supermix (Bio-Rad). The PCR amplification was performed for 44
cycles, each lasting 20 s at 95℃ and 65℃, and 30 s at 72℃, with initial
denaturation at 95℃ for 3 min. Cytokine mRNA levels were compared after
correction using the β-actin mRNA level as an internal standard. Expression
level was analyzed by gene expression analysis using the Chromo 4 instrument
(Bio-Rad Laboratories Inc., Hercules, CA, USA).
Inc., Cincinnati, OH, USA). RNA concentrations were measured from the absorbance
at 260 and 280 nm. Complementary DNA (cDNA) synthesis was performed using a
Maxime RT PreMix Kit (Intron Biotechnology, Seoul, South Korea), in accordance
with the manufacturer’s protocol. The following primers were used for real-time
RT-PCR analyses: IL-6, 5′-GTA CTC CAG AAG ACC AGA GG-3′ and 5′-TGC TGG TGA CAA
CCA CGG CC-3′; IL-1β, 5′-CAG GAT GAC ATG AGC ACC-3′ and 5′-CTC TGC AGA CTC AAA
CTC CAC-3′; TNF-α, 5′-TTG ACC TCA GCG CTG AGT TG-3′ and 5′-CCT GTA GCC CAC GTC
GTA GC-3′; and β-actin, 5′-AGG CTG TGC TGT CCC TGT AT-3′ and 5′-ACC CAA GAA GGA
AGG CTG GA-3′. The levels of IL-1β, IL-6, TNF-α, and β-actin mRNAs were measured
using a Chromo 4TM Real-Time PCR Detector (Bio-Rad Laboratories Inc., Hercules,
CA, USA). For relative quantification, the reactions were performed using 2×
iQTM SYBR Green Supermix (Bio-Rad). The PCR amplification was performed for 44
cycles, each lasting 20 s at 95℃ and 65℃, and 30 s at 72℃, with initial
denaturation at 95℃ for 3 min. Cytokine mRNA levels were compared after
correction using the β-actin mRNA level as an internal standard. Expression
level was analyzed by gene expression analysis using the Chromo 4 instrument
(Bio-Rad Laboratories Inc., Hercules, CA, USA).