Abi viia7 real time pcr detection system

Lung tissue was homogenized and snap-frozen in RLT buffer (Qiagen, Valencia, CA). Total RNA was extracted from lung tissue using the Qiagen RNeasy Mini kit (Qiagen). RNA was converted to cDNA using ABI reverse transcription reagents (ABI/Thermo Fisher Scientific, Carlsbad, CA) using a Bio Rad DNA Engine Thermal Cycler (Bio Rad, Hercules, CA). cDNA was then amplified using TaqMan reagents on the ABI Viia7 Real-Time PCR detection system (Thermo Fisher Scientific). The primer and probe sequences for murine glyceraldehyde 3-phosphate dehydrogenase (Gapdh) and Tnfa were previously published^{55 (link)}. The primers and probes for murine Arg1, Ccl2, Ccl7, and Ccl12 were purchased commercially (ABI Biosystems). The primer and probe sequences for Tgfb1 are forward 5′-TGACGTCACTGGAGTTGTACGG-3′, reverse 5′- GGTTCATGTCATGGATGGTGC-3′, probe 5′ - /56-FAM/TTC AGC GCT CACTGC TCT TGT GAC AG/3BHQ_1/ - 3’. Fold increase was determined over uninfected controls relative to Gapdh expression using the ∆∆CT calculation as previous described^{55 (link)}.

Freshly dissected tissue from the rim of healing appendages were manually dissociated with scissors, collected in TRI Regeant (Zymo) and then mechanically disrupted (Fastprep 24, Lysing Matrix D, MP Bio). Total RNA was isolated by Direct-Zol RNA MicroPrep (Zymo). RNA concentration was measured by Nanodrop 1000 (Thermo Scientific). cDNA synthesis was performed with Maxima Reverse Transcriptase (Thermo Scientific) or Superscript IV VILO (Thermo Scientific) per manufacturer’s instructions. One-step quantitative RT–PCR was performed and analyzed using an ABI ViiA7 Real-Time PCR detection system (Applied Biosystems) with TaqMan one-step RT–PCR Master Mix Reagents. Taqman probes were purchased for mouse Il-23r, beta-actin, and Trpa1 (Applied Biosystems).