Qpcr master mix

Total RNA of lung tissues from the above treated mice was extracted via Invitrogen Trizol (Carlsbad, USA) following the protocol of manufacturer. 1 μg RNA was reversely transcribed into cDNA by using the M-MLV reverse transcriptase (Promega, Madison, USA). The Bio-Rad qPCR Master Mix (Hercules, USA) was used to conduct qRT-PCR on the 7500 Real-Time PCR system. GAPDH were used to normalize the expression level of NOD1 and TLR4. The relative expression levels of NOD1 and TLR4 were calculated by using the 2^−ΔΔCT method. Primers were listed below: NOD1: Forward: 5’-AGGAGGCCAACAGACGCC-3’, Reverse: 5’-CTGACCTAGAGGGTATCG-3’; TLR4: Forward: 5’-AGCTCCTGACCTTGGTCTTG-3’, Reverse: 5’- CGCAGGGGAACTCAATGAGG-3’. GAPDH: Forward: 5’-CATCACTGCCACCCAG AAG ACTG-3’, Reverse: 5’-ATGCCAGTGAGCTTCCCGTTCAG-3’.

TRIzol Reagent (Sigma-Aldrich; Merck KGaA) was used for total RNA extraction and cDNA was synthesised using iScript cDNA Synthesis kit (Bio-Rad Laboratories, Inc.). REDTaq ReadyMix PCR reaction mix (primer sequences presented in Table I) was utilised for PCR under the following cycling conditions: 95°C for 5 min, followed by 36 cycles at 95°C for 30 sec, 55°C for 30 sec, 72°C for 40 sec and a final extension at 72°C for 10 min.
qPCR of BC cell cDNA samples was performed using the Icycler IQ5 system (Bio-Rad Laboratories, Inc.), the Amplifluor system (Intergen, Inc.) and qPCR Mastermix (Bio-Rad Laboratories, Inc.) to identify Psoriasin transcript expression, along with standards and negative controls. Psoriasin primers were designed using Beacon design software (Premier Biosoft International), with additional Z sequence (5′-ACTGAACCTGACCGTACA) on the reverse primer complementary to the universal Z probe (Intergen, Inc.). Reaction conditions were as follows: 95°C for 12 min, followed by 90 cycles at 95°C for 15 sec, 55°C for 40 sec and 72°C for 20 sec.