Suprimecript rt premix

Cells were homogenized with Trizol reagent (Invitrogen; Carlsbad, CA, USA) and shaken vigorously with chloroform for 15 min. Aqueous phases were then transferred to fresh tubes, isopropanol was added, incubated for 15 min at 4 °C, and centrifuged for 15 min at 12,000 rpm. After removing supernatants, pellets were washed with 75% ethanol and centrifuged for 5 min at 8000 rpm. The RNA pellets obtained were dried and dissolved in diethylpyrocarbonate water, and mRNA concentrations were calculated. mRNA was reverse transcribed to cDNA using SuPrimeCript RT Premix (Genetbio Inc.; Daejeon, Korea), and real-time PCR was performed using SYBR green master mix (BIOLINE; Taunton, MA, USA) and the CFX Connect System (Bio-Rad Inc.; Hercules, CA, USA). The primer sequences used were as follows: tumor necrosis factor-α (TNF-α) (NM_012675.3) (5′-ATT GCT CTG TGA GGC GAC TG-3′ and 5′-GGG GCT CTG AGG AGT AGA CG-3′); interleukin (IL)-6 (NM_012589.2) (5′-TCA TTC TGT CTC GAG CCC AC-3′ and 5′-GAA GTA GGG AAG GCA GTG GC-3′); IL-1β (NM_031512.2) (5′-AAA ATG CCT CGT GCT GTC TG-3′ and 5′-CCA CAG GGA TTT TGT CGT TG-3′); GAPDH (NM_017008.4) (5′-AGA CAG CCC CAT CTT GT-3′ and 5′-ACG GTG AGT CTT CTG ACA CC-3′).

Cells were homogenized with Trizol reagent (Invitrogen; Carlsbad, CA, USA), chloroform was added, and shaken vigorously for 15 min. The aqueous phase was then transferred to fresh tubes, isopropanol was added, incubated for 15 min at 4 °C, and centrifuged for 15 min at 12,000 g. Supernatants were removed and pellets were washed with 75% ethanol and centrifuged for 5 min at 8000 g. The RNA pellets obtained were dried and dissolved in diethyl pyrocarbonate water, and mRNA concentrations were calculated. mRNA was reverse transcribed to cDNA using SuPrimeCript RT Premix (Genetbio Inc.; Daejeon, South Korea). Real-time PCR analysis was performed using SYBR green master mix (BIOLINE; Taunton, MA, USA) and the CFX Connect System (Bio-rad Inc.; Hercules, CA, USA). Primer sequences were as follows; IL-1β (5′-AAA ATG CCT CGT GCT GTC TG-3′ and 5′-CCA CAG GGA TTT TGT CGT TG-3′); IL-6 (5′-TCA TTC TGT CTC GAG CCC AC-3′ and 5′-GAA GTA GGG AAG GCA GTG GC-3′); CCL2 (5′-AGC ATC CAC GTG CTG TCT C-3′ and 5′-GAT CAT CTT GCC AGT GAA TGA G-3′); TNF-α (5′-ATT GCT CTG TGA GGC GAC TG-3′ and 5′-GGG GCT CTG AGG AGT AGA CG-3′); GAPDH (5′-AGA CAG CCC CAT CTT CTT GT-3′ and 5′-ACG GTG AGT CTT CTG ACA CC-3′).