Chamq universal sybr qpcr

To extract the total RNA, a kit of EZBioscience (Roseville, US) was employed; then, we measured RNA concentration and purity by Nanodrop (Thermofisher) and was reversed to complementary DNA (cDNA) by HiScript III-RT SuperMix for qPCR (+gDNA wiper) (Vazyme, Nanjing, China). Subsequently, we conducted qPCR by ChamQ Universal SYBR qPCR (Vazyme, Nanjing, China) and detected by 7500 Real-Time PCR System (Thermofisher, US); the relevant gene expression was calculated via 2^−ΔΔCT. COX10-AS1 and GAPDH primers were synthesized by Sangon Biotech. The COX10-AS1 sequence: 5′-TATCGAACGGTACTTGCTTACG-3′ (F), 5′-TGGCTAGTGACCCGGTAGTCA-3′ (R); CASP1 sequence: 5′-TTTCCGCAAGGTTCGATTTTCA-3′ (F), 5′-GGCATCTGCGCTCTACCATC-3′ (R); NLRP3 sequence: 5′-GATCTTCGCTGCGATCAACAG-3′ (F), 5′-CGTGCATTATCTGAACCCCAC-3′ (R); GAPDH sequence: 5′-GAAGGTGAAGGTCGGAGTC-3′ (F), 5′-GAAGATGGTGATGGGATT TC-3′ (R).

Total RNAs were prepared using a Gene JET RNA Purification Kit (k0731, Thermo Scientific). cDNAs were prepared with a HiScript II Reverse Transcriptase (Glycerol-free) (RL201-01, Vazyme Biotech) and processed for RT-qPCR (Cham Q Universal SYBR qPCR, Vazyme Biotech) using the primers listed in Supplementary Table.

Real-time quantitative polymerase chain reaction (RT-qPCR) analysis was carried out to determine the gene expression of ECFCs and PBMCs. Briefly, the various surfaces were washed in ice-cold PBS and lysed with TRIzol (R401-01, Vazyme, China) for the subsequent total RNA isolation after the ECFCs/PBMCs were captured and cultured for 4 and 8 weeks. Then, the sequence amplification kit (R323, Vazyme, China) was used to apply the one-step mRNA reverse transcription. The obtained cDNA was adopted as the template for subsequent PCR amplification. The RT-qPCR reactions were performed using the CFX96 system (BioRad, United States) and the ChamQ Universal SYBR qPCR (Q711, Vazyme, China). The expression of the target gene was calculated according to the following equation: $\mathrm{F}\mathrm{o}\mathrm{l}\mathrm{d}\mathrm{c}\mathrm{h}\mathrm{a}\mathrm{n}\mathrm{g}\mathrm{e}=2^{\left(-\Delta \Delta \mathrm{C}\mathrm{t}\right)}.$
Reduced glyceraldehyde-phosphate dehydrogenase (GAPDH) expression was used as the reference gene. The primers used were designed by Primer Premier 5 (PREMIER Biosoft, Canada), and the details are given in Supplementary Table S1.