Myocardial tissue samples were homogenized in TRI Reagent (1 mL), and 200 μL chloroform was added. The sample was centrifuged at 12 000 g (4°C, 15 min), and the supernatant was transferred to a sterile centrifuge tube. Isopropanol (500 μL) was added to the supernatant, mixed, and then centrifuged again at 12 000 g (4°C, 10 min). The upper layer of transparent liquid was removed and 1 mL of 75% ethanol was added. This step was repeated 5 to 10 times. After the final ethanol wash, the sample was centrifuged at 7600 g (4°C, 5 min), placed at room temperature for 3 to 5 min, air dried, and dissolved in RNase-free water (30 μL).
mRNA was reverse transcribed to synthesize cDNA, followed by PCR. Each PCR assay used 1 μg RNA per 20 μL reaction. The experiment was performed according to the manufacturer’s instruction (PCR array kit: Mouse Mitochondrial Energy Metabolism, SABiosciences, Qiagen, PAMM-008Z). Briefly, every reaction was performed using 10 ng cDNA per 25 μL reaction volume. Quantitative PCR was performed under the following conditions: 95°C 10 min; 40 cycles of 95°C, 15 sec and 60°C, 1 min. The assays were performed on a Bio-Rad CFX ConnectTM Real-Time PCR Detection System. The qPCR master mix (QIAGEN, 330 523) was used for qPCR assay.
mRNA was reverse transcribed to synthesize cDNA, followed by PCR. Each PCR assay used 1 μg RNA per 20 μL reaction. The experiment was performed according to the manufacturer’s instruction (PCR array kit: Mouse Mitochondrial Energy Metabolism, SABiosciences, Qiagen, PAMM-008Z). Briefly, every reaction was performed using 10 ng cDNA per 25 μL reaction volume. Quantitative PCR was performed under the following conditions: 95°C 10 min; 40 cycles of 95°C, 15 sec and 60°C, 1 min. The assays were performed on a Bio-Rad CFX ConnectTM Real-Time PCR Detection System. The qPCR master mix (QIAGEN, 330 523) was used for qPCR assay.