Cardiac Gene Expression Analysis

Total RNA was extracted from cardiomyocytes or H9c2 cells using TRIzol reagent (Invitrogen), according to the manufacturer’s instructions. EasyScript cDNA Synthesis SuperMix (TransGen Biotech, Beijing, China) was used to synthesize cDNA from mRNA, and TransScript^® miRNA First-Strand cDNA Synthesis SuperMix (TransGen Biotech) was used to synthesize cDNA from miRNA. Quantitative PCR (qPCR) was performed using TransStart Top Green qPCR SuperMix (TransGen Biotech). mRNA levels were normalized to that of actin, and miRNA levels were normalized to that of U6, using a delta-delta-Ct method as previously described.^{36 ,37 (link)} All experiments were independently performed in triplicate. The following primers were used: 5’-CTCACTAAAGGCACCGAAGG- 3’ and 5’-GGCAGAGAAGTTGCTTGTGG- 3’ (MALAT1); 5’-CTTCGGGGGTAGGATTGAC- 3’ and 5’-CTTGGGATCTTTTGCGATCT-3’ (ANP); 5’-TGATTCTGCTCCTGCTTTTCC-3’ and 5’-TTCTTTTGTAGGGCCTTGGTC- 3’ (BNP); 5’-CGCCTGTCAGCTTGTAAATG- 3’ and 5’-ACAACCCCTACGATTATGCG-3’ (β- MHC); 5’-TCCTGGTAGGCCAACAGGCT-3’ and 5’-AGCTAATGTTGAGCTGCACTTG- 3’ (HMGB2); 5’-CCGCATCTTCTTGTGCAGTG- 3’ and 5’-GAGAAGGCAGCCCTGGTAAC- 3’ (GAPDH); 5’-GGGAACATTCAACGCTGTCG-3’ and 5’-GTGCGTGTCGTGGAGTCG-3’ (miR-181a); and 5’- CTCGCTTCGGCAGCACA-3’ and 5’-AACGCTTCACGAATTTGCGT- 3’ (U6).