Primescript tm 1st strand cdna synthesis kits

Total RNA was extracted from WISH cells using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). Total mRNA (1 µg) was synthesized to cDNA using oligo(dT) PrimeScriptTM 1st strand cDNA Synthesis Kits (TaKaRa Bio, Shiga, Japan and Clontech, BD Biosciences, Palo Alto, CA, USA) according to the manufacturer's instructions. RT-PCR was done on a SimpliAmp Thermal Cycler (Applied Biosystems, Foster City, CA, USA). PCR primers used were: IL-1β, Forward: 5′- CTC GCC AGT GAA ATG GCT - 3′, Reverse: 5′ - GTC GGA GAT TCG TAG CTG GAT - 3′; TNF-α, Forward: 5′ - CCA GGC AGT CAG ATC TTC - 3′, Reverse: 5′ - GTT ATC TCT CAG CTC CAC GC - 3′; and β-actin, Forward: 5′ - GAC CTG ACT GAC TAC CTC ATG - 3′, Reverse: 5′ - CGC TCA TTG CCA ATG GTG ATG - 3′. Amplification of interleukin (IL)-1β / β-actin was performed using 35 cycles of 95℃ for 30 s, 55℃ for 30 s, and 72℃ for 30 s with a final extension of product at 72℃ for 7 min. Amplification of tumor necrosis factor-alpha (TNF-α) / β-actin was performed using 35 cycles of 94℃ for 30 s, 54℃ for 30 s, and 72℃ for 30 s with a final extension of product at 72℃ for 10 min. PCR-amplified products were separated on a 1.5% stained agarose gels. The Gel Doc ImageQuant LAS 500 System (GE Healthcare Bio-Sciences AB, Uppsala, Sweden) was used to assay the PCR products. β-actin was used to normalize all target genes. The data were analyzed using the Image J program.