Iq5 pcr instrument

Frozen vascular tissue (40–60 mg) was taken, from which all RNAs were extracted and synthesized into cDNA by reverse transcription. The primer sequences of NLPR3 were 5′-GCTAAGAAGGACCAGCCAGA-3′ and 5′-CCAGCAAACCTATCCACTCC-3′. The primer sequences of GAPDH were 5′-GCTAAGAAGGACCAGCCAGA-3′ and 5′-CCAGCAAACCTATCCACTCC-3′. Quantitative real-time PCR was performed using a two-step amplification method, and all information was collected by the IQ5 PCR instrument (Bio-Rad, Hercules, CA, USA). As the GAPDH housekeeping gene was used as an internal control, we calculated the transcriptional levels of target genes using the 2^−△△Ct method based on cycle threshold (Ct) values.

The qMSP was performed as described by Yan et al. [43 (link)]. Bisulfite-converted genomic DNA was subject to real-time PCR with methylation-specific primers (Additional file 1: Table S1). A SYBR Green I PCR Kit (Toyobo) was used to conduct qMSP in an iQ5 PCR instrument (Bio-Rad). After reactions, analysis of melting temperature was performed to ensure that a specific amplicon was generated. Col2A1 (NM_033150) was used for standard curve construction and as a loading control. Methylation percentage was calculated as: (mean of target gene)/(mean of Col2A1). Fold change was calculated as: (targeted DNA methylation percentage)/(mock methylation percentage).