Prism 7500

Total RNA was isolated from cultured cells with RNAiso Plus (Takara, Dalian, China), and cDNA was synthesized with the PrimeScript RT Reagent Kit (Takara). Then, 2 μl of cDNA was used for real-time PCR reactions in a Prism 7500 real-time thermocycler (Applied BioSystems, Foster City, CA, USA) with SYBR Green Ex Taq (Takara) according to the manufacturer's instructions. The primer sequences are provided in Supplementary Table 10. Each group was made in triplicate.

Total RNA was prepared using the RNeasy mini kit as described above. RT-PCR was performed using specific primers and One-Step SYBR Green PCR mix (Takara), according to the manufacturer's manual. qRT-PCR was performed using a Prism 7500 sequence detection system (Applied Biosystems). Samples were run in triplicate and all data were normalized to GAPDH mRNA expression as an internal control.

Total RNA was extracted from the R28 cells using TRIzol reagent (Invitrogen, Waltham, MA, USA), and cDNA was synthesized with a Hifair III 1st Strand cDNA Synthesis Kit (Yeasen Biotechnology, Shanghai, China). Quantitative real-time polymerase chain reaction (RT-PCR) was performed using a Hieff qPCR SYBR Green Master Mix (Low Rox, Yeasen Biotechnology, Shanghai, China) with a sequence detection system (Prism 7500, Applied Biosystems, Waltham, MA, USA) according to the manufacturer’s instructions. The specific primers were designed by Sangon Biotech (Shanghai, China), including TNF-α (forward 5′-ACCATGAGCACGGAAAGCAT-3′ and reverse 5′-AACTGATGAGAGGGAGCCCA-3′), IL-6 (forward 5′-TCTGGTCTTCTGGAGTTCCGT-3′ and reverse 5′-CTTGGTCCTTAGCCACTCCT-3′), IL-1β (forward 5′-TACCTATGTCTTGCCCGTGG-3′ and reverse 5′-TAGCAGGTCGTCATCATCCC-3′), and GAPDH (forward 5′-AGTGCCAGCCTCGTCTCATA-3′ and reverse 5′-TGAACTTGCCGTGGGTAGAG-3′). Relative mRNA levels were normalized to those of GAPDH and were calculated using the 2^−ΔΔCT method. Each sample was measured in triplicate wells, and the experiments were repeated three times.

After all experimental procedures, isolated cerebral samples were homogenized, isolating RNA using the trizol reagent (Invitrogen) as previously described by us.
⁴⁰ (link) Total RNA was then converted into cDNA using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems). The quantification of gene expression was determined by Prism 7500 real‐time PCR (Applied Biosystems). All reactions were performed under the following conditions: 95°C for 15 min, 40 cycles of 95°C for 10 s, and 60°C for 30 s. β‐actin was used as the control gene, and all data are represented as relative mRNA expression on gene expression. The primers for rat NLRP3, C‐terminal caspase recruitment domain (ASC), IL‐1β, IL‐18, GTPase‐activating protein‐binding protein 1 (G3BP1), T cell‐restricted intracellular antigen‐1 (TIA1), DDX3X, synaptophysin (SYN), post‐synaptic density protein‐95 (PSD‐95), and brain‐derived neurotrophic factor (BDNF), vascular endothelial growth factor (VEGF), angiopoietin‐1 (Ang‐1), and angiopoietin‐2 (Ang‐2), and β‐actin are shown in Table 1.

mRNA expression was analyzed in triplicate by RT-qPCR on the ABI Prism7500 sequence detection system using predesigned assay-on-demand primers and probes (Applied Biosystems). Hypoxanthine-guanine phosphoribosyltransferase (HPRT1 Hs01003267_m1) was used as an internal control and mRNA expression levels were given as fraction of mRNA levels in control cells. Primers and probes used were: CHOP (Hs01090850_m1), FLIP(L) (AIN1EV0), FLIP(S) (Ss03391532_m1), TRAIL-R2 (Hs00366278_m1), GOT1 (Hs00157798_m1), GOT2 (Hs00905827_g1) and GPT2 (Hs00370287_m1).

mRNA expression was analyzed in triplicate by reverse transcriptase-qPCR on the ABI Prism7500 sequence detection system using predesigned assay-on-demand primers and probes (Applied Biosystems). Hypoxanthine-guanine phosphoribosyltransferase (HPRT1 Hs01003267_m1) was used as an internal control and mRNA expression levels of ATF6, TRAIL, and Puma were given as fraction of mRNA levels in control cells. Primers and probes used were: ATF6 (Hs00232586_m1), TRAIL (Hs00921974_m1), and Puma (Hs0024075_m1).