One step rt pcr universal master mix reagent

Total RNA was isolated from tissues and cells using the RNeasy Mini Kit and RNase-Free DNase Kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s instructions, with deoxyribonuclease (DNase) treatment performed on the column. RNA purity and yield were assessed by evaluating the A260/A280 ratio and concentration using a NanoDrop One Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). The One-Step RT–PCR Universal Master Mix reagent (Thermo Fisher Scientific) was used to quantify the target gene mRNA levels. Quantitative real-time polymerase chain reaction (qRT–PCR) was performed with the CFX96 Real-Time System C1000 Touch Thermal Cycler (Bio-Rad) using predesigned primer–probe sets (Thermo Fisher Scientific) for β-myosin heavy chain (β-Mhc) (Myh7b, Mm01249941_m1), skeletal muscle α-actin (Ska) (Acta1, Mm00808218_g1), brain natriuretic peptide (Nppb) (Mm01255770_g1), interleukin-6 (Il-6) (Mm00446190_m1), lipocalin 2 (Lcn-2) (Mm01324470_m1), prostaglandin-endoperoxide synthase 2 (Ptgs2) (Mm00478374_m1), and the reference gene peptidylprolyl isomerase B (PPIB). The thermocycling parameters for each reaction were 48 °C for 30 min and 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 60 s. Values measured for each primer–probe set were normalized to PPIB.