Rna easy

700 µL of RNA-easy (Vazyme, China) was added to tumor tissues (30 mg), and the tissues were ground (40 m/s, 20 s, twice) using a homogenizer (MP Biomedicals, USA). The homogenate was centrifuged at 13,000 rpm at 4 °C for 10 min, and the supernatant was pipetted into 2 mL EP tubes. The RNA was extracted according to the RNA-easy instructions, and then cDNA was obtained by reverse transcription according to the instructions of HiScript III All-in-one RT SuperMix Perfect for qPCR (Vazyme, China). qPCR was performed by using Ssofast EvaGreen Supermix (Biorad, USA), and the relative expression level of each gene was calculated according to 2^−∆∆Ct. Information on the primers is shown in Supplementary material: Table S4 (Beijing Tsingke Biotech Co., Ltd.)

Cells were grouped and treated as in Section 2.5. Then, the expressions of NLRP3 pathway-relative factors, TNF-α and TGF-β, were measured using quantitative real-time PCR. We also determined gene expression in cells treated with LPS and nanoparticles for 12 h as well as nanoparticles alone for 24 h.
The total RNA of RAW264.7 was extracted by using RNA-Easy (Vazyme). RNA was reverse-transcribed into cDNA using a reverse transcription kit (ABScript III RT Master Mix for qPCR with gDNA Remover, ABclomal). RT-qPCR was performed using Universal SYBR Green Mix (ABclomal), cDNA, and primers under the following conditions: 95°C for 5 s and 60°C for 30 s with 40 cycles. β-Actin was an internal control of genes. Data results were analyzed with 2^−ΔΔCt. The primer sequences were as shown in Table 1.