Next sybr qpcr mix

Total RNA was extracted from lung tissue or RAW264 cells using an RNeasy kit in accordance with the manufacturer’s protocol. The samples were reverse-transcribed using the PrimeScript RT master mix described above. The synthesized cDNA was used in the real-time PCR experiments with THUNDERBIRD Next SYBR qPCR mix and analyzed with a Bio-Rad (Hercules, CA, USA) CFX96™ real-time system and CFX Manager™ software. Specificity was confirmed by electrophoretic analysis of the reaction products and the inclusion of template- or reverse-transcriptase-free controls. To normalize the amount of total RNA present in each reaction, glyceraldehyde-3-phosphate dehydrogenase (Gapdh) cDNA was used as an internal standard. Primers were designed using the Primer-BLAST website (https://www.ncbi.nlm.nih.gov/tools/primer-blast/, accessed on 4 July 2022). Primer sequences are described in Supplementary Figure S1.

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)].

Total RNA was extracted from GT1-7 cells using FastGene™ RNA Basic kit in accordance with the manufacturer’s protocol. The samples were reverse-transcribed using the PrimeScript RT master mix, and the resulting cDNA was used in the real-time PCR experiments with THUNDERBIRD Next SYBR qPCR mix, and analyzed with a Bio-Rad (Hercules, CA, USA) CFX96™ real-time system and CFX Manager™ software (Version 3.1). Specificity was confirmed by electrophoretic analysis of the reaction products and the inclusion of template- or reverse-transcriptase-free controls. To normalize the amount of total RNA present in each reaction, glyceraldehyde-3-phosphate dehydrogenase (Gapdh) cDNA was used as an internal standard. Primers were designed using the Primer-BLAST website (https://www.ncbi.nlm.nih.gov/tools/primer-blast/, accessed on 1 April 2023). Primer sequences will be provided upon request.