Quantifying Inflammatory Markers in Mice

The expression levels of inflammatory markers in the ears of the mice were measured using qRT-PCR. Total RNA in the lymph nodes and skin tissue was isolated using Trizol^® (Invitrogen, Waltham, MA, USA). The RNA was reverse transcribed into cDNA using MMLV reverse transcriptase (Promega, Madison, WI, USA). Thunderbird Next SYBR qPCR Mix was used for qRT-PCR on a CFX Connect Real-Time System (Bio-Rad, Hercules, CA, USA). The PCR program consisted of a cycle at 95 °C for 4 min, 40 cycles at 95 °C for 30 s, 57 °C for 30 s, and the last at 95 °C for 30 s. With the help of CFX Maestro Software version 2.3 (Bio-Rad Laboratories, Hercules, CA, USA), the obtained data were analyzed using the 2^−∆∆Ct method. The primer sequences are listed in Table 1. The results were normalized to GAPDH gene expression levels [35 (link)].