Cfx96 c1000 touch thermal cycler multicolor real time pcr detection system

Total RNA was isolated from the GM tissue homogenate using Trizol™ (Invitrogen, Carlsbad, CA) and reverse transcribed into cDNA using the Superscript™ III First Strand Synthesis system (Invitrogen, Carlsbad, CA). Following cDNA synthesis, quantitative real‐time polymerase chain reaction (PCR) was performed using CFX96 C1000 Touch™ Thermal Cycler Multicolor Real‐Time PCR Detection System (Bio‐Rad, Hercules, CA) with SYBR Green (Invitrogen, Carlsbad, CA). PCR was carried out for pyroptotic initiator HMGB1, inflammasome marker NLRP3, pyroptotic markers (caspase‐1, IL‐1β, IL‐18, and GSDMD), and for muscle atrophy marker muscle RING‐finger protein‐1 (MuRF1). The used list of mouse‐specific primers is presented in TableS1B. Glyceraldehyde 3‐phosphate dehydrogenase was used as loading control. Quantitative PCR was performed with an initial step of denaturation at 50°C for 2 min, 95°C for 10 min, followed by 40 cycles of 95°C for 20 s and 60°C for 20 s. Melt curves were established and normalized fold expression was calculated by using 2^−∆∆Ct method.