Quantitative Real-time PCR Analysis of Gene Expression

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.