Determination of IL-18 mRNA expression by reverse transcription-quantitative PCR

Total RNA was extracted from A549 cells using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.). Total RNA (1 µg) was reverse transcribed to cDNA using a High Capacity cDNA Reverse Transcription kit (Thermo Fisher Scientific, Inc.) in accordance with the manufacturer's protocol. qPCR was performed using the SYBR Green Reverse Transcription PCR Master mix (Takara Biotechnology Co., Ltd.) on an Applied Biosystems 7300 plus reverse transcription PCR system (Thermo Fisher Scientific, Inc.). The sequences were as follows: IL-18 forward, 5′-ACTGTACAACCGCAGTAATAC-3′ and reverse, 5′-AGTGAACATTACAGATTTATCCC-3′ (product size, 434 bp); β-actin forward, 5′-CTCCATCCTGGCCTCDCTGT-3′ and reverse, 5′-GCTGTCACCTTCACCGTTCC-3′. The following thermocycling conditions were used for qPCR: Initial denaturation at 95°C for 30 sec; 40 cycles of 95°C for 5 sec and 60°C for 30 sec. Expression levels were quantified using the 2^−∆∆Cq method (12) and β-actin was used as an internal reference gene.