2.11. Quantitative Real-Time Polymerase Chain Reaction (PCR) Analysis for Cytokine Gene Expression

Quantitative real-time PCR assessed the relative quantities of mRNA for IL-4, IL-13, TNF-α, and IL-1β. Total RNA molecules were isolated from frozen lung tissues using RNA Bee solution (Tet-Test Inc., Friendswood, TX, USA). After quantification of the RNA concentration, the complement DNA (cDNA) was synthesized using a mixture of oligo-dT primer (Invitrogen, Carlsbad, CA, USA), dNTP and reverse transcriptase (Superscript II, 18064-014, Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA). Q-PCR was then conducted using a cDNA template and 2× Power SYBR Green (TOYOBO Co., Osaka, Japan) as described in previous studies [26]. The reaction cycle at which PCR products exceeded this fluorescence intensity threshold during the exponential phase of PCR amplification was considered as the threshold cycle (CT).