Cfx manager 3.2

RNA was extracted with Trizol reagent (Invitrogen). cDNA was synthesized with 1 µg of total RNA with iScript Reverse Transcription Supermix (Biorad, USA). Gene expression was determined by qRT-PCR (Biorad, USA) using SYBR Green Supermix with gene-specific primer sets real-time PCR using primers as previously used^{3 (link),47 (link),48 (link)}. The common endogenous reference genes, such as 18 s rRNA, Gapdh, Actb, 36B4, B2M, Tbp, and Hprt were evaluated using the comparative delta-Ct method^{49 (link)} as well as M-value, the reference gene expression stability using Bio-Rad CFX Manager 3.2. (Biorad, USA). Hprt was most stable among groups, so mRNA levels were normalized to Hprt using the cycle threshold (ΔΔCT), and relative fold changes were reported compared to the CON. Primers used for this study are listed in Supplementary Table 2.

Cardiac gene expression was determined according to the protocol described previously [10 (link)]. The following genes were determined: (1) gene-associated pathological cardiac hypertrophy (α-myosin heavy chain (Myh6), beta-myosin heavy chain (Myh7), phospholamban (Pln), sarcoplasmic reticulum Ca2+ ATPase 2a (Atp2a2), α-skeletal actin (Acta1), atrial natriuretic peptide (Nppa), and brain natriuretic peptide (Nppb)); (2) interstitial collagen content collagen type 1 alpha chain (Col1a1, Col3a1, and Col8a1); (3) Ccl2, also known as MCP-1, an important proinflammatory chemokine; (4) glucose transport (Glut4 and Irs-1); (5) gluconeogenesis (Pepck), fatty acid transport and oxidation (Cd36, Cpt 1, Cpt2, and Mcad); (6) genes regulating mitochondrial biogenesis (Ppargc1a, Tfam, Nrf1, Nrf2); and (7) genes regulating mitochondrial dynamics (Drp1, Mfn1, Mfn2, Opa1, Cypd). The common reference genes, such as Gapdh, Actb, 36B4, B2M, Tbp, and Hprt, were evaluated using M-value, the reference gene expression stability using Bio-Rad CFX Manager 3.2. (Biorad, Hercules, CA, USA). B2M was most stable among groups, so mRNA levels were normalized to B2M using the cycle threshold (ΔΔCT), and relative fold changes were reported compared to the LL. Primers used for this study are listed in Table S1.