Mm00840904 m1

RNA from the aorta was isolated using the RNeasy Mini Kit according to the manufacturer’s protocol (Qiagen GmbH, Hilden, Germany), followed by cDNA synthesis. To assess the mRNA expression of the target genes TNF-α, VCAM-1, CD4, CD8, NOD2, NLRP3, collagen I and III, real-time PCR (Eppendorf Mastercycler epgradient realplex, Hamburg, Germany) was performed using gene expression assays for TNF-α Mm00443258_m1, VCAM-1 Mm01320970_m1, CD4 Mm00442754_m1, and CD8a Mm01182107_g1, nucleotide-binding oligomerization domain containing (NOD) 2 Mm00467543_m1, nucleotide-binding domain, leucine-rich-containing family, pyrin domain-containing (NLRP) 3 Mm00840904_m1, Col1a1 Hs00164004_m1, and Col3a1 Hs00943809_m1 from Applied Biosystems, respectively. mRNA expression was normalized to the housekeeping gene CDKN1b (gene expression assay Hs00153277_m1) and relatively expressed with the control group set as 1.

Total RNA was extracted from frozen NRCMs, cardiac fibroblasts and frozen heart tissue using TRIzol reagent (Invitrogen), and quantitative real-time PCR was performed using a StepOnePlus Real-time PCR System and the StepOne Software program (Applied Biosystems), as previously described.⁷ (link) The RT‒PCR protocol consisted of one cycle at 95°C for 20 s followed by 40 cycles at 95°C for 1 s and 60°C for 20 s using the primers for URAT1 (Applied Biosystems, Mm01244861_m1 and Rn01479630_g1), TNFα (Applied Biosystems, Mm00443258_m1 and Rn99999017_m1), MCP1 (Applied Biosystems, Mm00441242_m1 and Rn00580555_m1), IL-1β (Applied Biosystems, Mm00434228_m1 and Rn00580432_m1), CD68 (Applied Biosystems, Mm00432403_m1), NLRP3 (Applied Biosystems, Mm00840904_m1) and GAPDH (Applied Biosystems, Mm03302249_g1 and Rn01775763_g1). The transcriptional levels were determined using the ΔΔCt method with normalization to GAPDH.