Tri reagent kit

Total RNA was isolated from liver tissues by using TRI Reagent Kit (Molecular Research Center, Inc., Cincinnati, OH, USA) and reverse transcribed into cDNA by using the StaRT Reverse Transcription Kit (AnyGenes, Paris, France). qRT-PCR was performed using a 7500 Fast Real Time PCR System thermal cycler (Applied Biosystems, MA, USA) according to the instruction of the TB Green^® Premix Ex Taq™ (Takara Bio Inc., Shiga, Japan). The related mRNA expression was normalized to 18s mRNA, and qRT-PCR data were analyzed using the comparative 2^-△△Ct method [34 (link)]. The following primers were used to amplify rat genes: forward (F) primer 5′-CCAGTGCCCTGCTTCATC-3′ and reverse (R) primer 5′GCAGGGCAAGTTAGGATCAG-3′ for eNOS, F primer 5′-CTTTGCCACGGACGAGAC-3′ and R primer 5′-TCATTGTACTCTGAGGGCTGAC-3′ for iNOS, F primer 5′-GTCAAGCACAGGGTGACAGA-3′ and R primer 5′-ATCACCTGCAGCTCCTCAAA-3′ for HO-1d, F primer 5′ TGAAAGCCTAGAAAGTCTGAAGAAC-3′ and R primer 5′-CGTGTTACCGTCCTTTTGC -3′ for IFN-γ, and F primer 5′- GGTGCATGGCCGTTCTTA-3′ and R primer 5′-TCGTTCGTTATCGGAATTAAC-3′ for 18S. The other rat primers (NLRP3, PYCARD, CASP1, IL-1β, IL-18, TNF-α) were custom primers and validated (AnyGenes, Paris, France).

Total RNA was extracted from unactivated or activated PBMCs (CD3 1 μg ml⁻¹ for 6 h) using a TRI Reagent Kit (Molecular Research Center Inc., Cincinnati, OH, USA), following the manufacturer's protocol. The integrity of intact total RNA was verified by measuring the ratio of rRNA OD (28S/18S) and the RNA integrity number with an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA). Genomic DNA was extracted from whole blood from the patient by using the PAXgene Blood DNA Kit (QIAGEN Inc., Valencia, CA, USA) following the manufacturer's protocol.