Rnaeasy total rna extraction kit

According to the manufacturer’s instructions, total RNA was extracted from left ventricular tissues of mice treated with or without cardiac I/R injury and SIN using the RNAeasy total RNA extraction kit (Qiagen, GmbH, Hilden, Germany). The cDNA was generated with the reverse transcription kit from Promega (Madison, WI, United States). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control. Real-time PCR was performed using a reaction system containing 1 × Faststart SYBR Green Master Mix (Roche Molecular Biochemicals), 2 μl diluted cDNA, 2 mM MgCl₂, and 0.5 μM primer. The experiment was repeated three times. Primers used in the experiments were cited as follows: TNF-α, 5′-CTG​TGA​AGG​GAA​TGG​GTG​TT-3′ (F), 5′-GGTCAC TGTCCCAGCATCTT-3′ (R); IL-β, 5′-TGGAAAAGC GGTTTGTC TTC-3′ (F), 5′-TAC​CAG​TTG​GGG​AAC​TCT​GC-3′ (R); IL-6: 5′-AGA​GAT​ACA​AAG​AAA​TGA​TGG​A-3′ (F), 5′-AGC​TAT​GGT​ACT​CCA​CAA-GAC​CA-3′ (R); GAPDH: 5′-GGC​CTT​CCG​TGT​TCC​TAC-3′ (F), 5′-TGT​CAT​CAT​ATC​TGG​CAG​GTT-3′ (R) (Huang et al., 2018 (link)).

RNA was extracted from kidneys using RNAeasy Total RNA extraction kit (Qiagen, Valencia, CA, USA) and quantified with a Nanodrop 2000 spectrophotometer (ThermoFisher Scientific, Waltham, MA, USA). Extracted RNA was converted to cDNA using the Applied Biosystems^TM High-Capacity cDNA Reverse Transcription Kit, as per manufacturer guidelines on the iCycler iQ5 (Bio-Rad, Hercules, CA, USA). Quantitative PCR with Taqman reagents was performed to quantify mRNA expression using primers purchased from Life Technologies (Carlsbad, CA, USA). Primers used were designed for: fibronectin 1 (Fn1; Mm01256744_m1), hydroxysteroid 11-beta dehydrogenase 2 (Hsd11b2; Mm01251104_m1), NADPH oxidase 4 (Nox4; Mm00479246_m1), serum/glucocorticoid regulated kinase 1 (Sgk1; Mm00441380_m1), sodium channel epithelial 1 subunit alpha (Scnn1α; Mm00803386_m1), and ubiquitin C (Ubc; Mm01198158_m1) as the reference gene.