2.7. Real-Time Quantitative RT-PCR

The expression of NLRP3, IL-1β, and caspase-1 mRNAs was determined by real-time quantitative reverse transcription polymerase chain reaction (RT-PCR). Briefly, total RNA was isolated from the ischemia core area by the TRIzol Reagent (Invitrogen, USA) and cDNA was synthesized by the GeneAmp PCR System 9700 (ABI, USA). Quantitative real-time PCR was performed by the SYBR Green kit (Takara, Japan) on a LightCycler480 System (Roche Diagnostics). The reaction was performed with a predegeneration step at 95°C for 30 s and annealing and extension at 60°C for 20 s for 40 cycles. The relative quantity of the target mRNA was normalized to the level of β-actin mRNA as the internal control. Table 1 describes the sequences of gene-specific primers used in this study. Primer sequences of targeted gene were used as previously described [13]. The relative fold change of NLRP3, IL-1β, and caspase-1 mRNAs was determined by the 2^−ΔΔCt method.

Sequences of primer used for real-time PCR assay (5′-3′).