Quantifying RAS and Senescence Genes

Total RNA was extracted from mouse tissues using Sepasol (R)‐RNA Super G (Nacalai Tesque, Kyoto, Japan). cDNA was synthesized using a ReverTra Ace quantitative real‐time polymerase chain reaction kit (FSQ‐101; Toyobo, Osaka, Japan) according to the manufacturer's instructions. We performed quantitative estimation of the genes associated with RAS (AT1, AT2, Mas, Renin, Angiotensinogen, ACE, and ACE2), the proinflammatory senescence‐associated secretory phenotype (p16, p19, p21, p53, PAI‐1, IGFBP2, MMP13, IL‐6, TNF‐α, and MCP‐1),²⁵ (link), ²⁶ (link), ²⁷ (link) muscle‐specific gene markers of senescence (MuRF‐1, Atrogin‐1, and Myostatin),²⁸ (link), ²⁹ (link) mitochondrial function (Mfn1, Mfn2, and DRP1), and fibrosis (α‐SMA, TGF‐β1, COL1A1, and CTGF), relative to GAPDH expression in the vastus muscle. Quantitative real‐time polymerase chain reaction was performed with the SYBR Green qPCR system (Takara, Shiga, Japan) and a 7900HT Fast Real‐Time PCR instrument (Applied Biosystems, Chiba, Japan). Data were analyzed with SDS software 2.4 (Applied Biosystems). Relative expression was calculated using the ΔΔCt method, with data normalized using GAPDH as an internal control. The primer pairs used in the study are listed in Table S1.