Total RNA was extracted from snap-frozen myocardial tissue using miRNeasy Mini kit (Qiagen) following the manufacturer’s instructions and then digested with DNase I (18068, Invitrogen). cDNA was synthesized with SuperScript III first strand (18068, Invitrogen) as previously described (25 (link), 26 (link)). Real-time PCR was performed using the Quantifast Sybrgreen PCR kit (204057, Qiagen) and the QuantStudio 3 Real-Time PCR System (A28567, Thermo Fisher). Ct values were normalized with respect to β2-microglobulin (β2M). Fold changes were calculated with respect to WT-N mice compared with different treatment groups. Fold changes were calculated with respect to HDAC1 when compared among different HDACs.
The following primer sets were used (forward, reverse):
β2M, 5′-
ATGTGAGGCGGGTGGAACTG , 5′-
CTCGGTGACCCTG GTCTTTCTG ; atrial natriuretic peptide (ANP), 5′-
GCCCTGAGTGAGCAGACTG , 5′-
GGAAGCTGTTGCAGCCTA ; HDAC1, 5′-
GTCCGGTGTTTGATGGCTTG , 5′-
GCAGTGGGTAGTTCACAGCA ; HDAC2, 5′-
TATCCCGCTCTGTGCCCTAC , 5′-
GAGGCTTCATGGGATGACCC ; HDAC3, 5′-
GACTTCTACCAGCCGACGTG , 5′-
GCTTCTGGCCTGCTGTAGTT ; HDAC8, 5′-
CTGGACATACTTGACCGGGG , 5′-
ACCGCTTGCATCAACACACT ; HDAC4, 5′-
GGGAGCAGCATCATGGTTCAA , 5′-
TGAGAACTGGTGGTCCAAGC ; HDAC5, 5′-
AGAGTGACGTCTCCGAATGTTG , 5′-
AGGAGTCCGTGGCAGGATTT ; HDAC6, 5′-
AGATCTGCGCGAGTGGAAG , 5′-
CTCTCTGATGGCATGGAGCC ; HDAC7, 5′-
TATTCCCTACAGCCTGCCCACT , 5′-
ACAGTGGGGCATGAGAGACT ; HDAC9, 5′-
CCATTGCCACGTGAACAACC , 5′-
GACGACAGGATCCACCACAG ; TGF-β, 5′-
GCCCGAAGCGGACTACTATG,5′-TTTGGGGCTGATCCCGTTG ; FN1, 5′-
AGAAGACAGGACAGGAAGCTC , 5′-
ATGGCGTAATGGGAAACCGT ; LOX, 5′-
TTCCAAGCTGGTTTCTCGCC , 5′-
GTCCGATGTCCCTTGGTTCT ; MMP9, 5′-
CGCTCATGTACCCGCTGTAT , 5′-
TGTCTGCCGGACTCAAAGAC .
The following primer sets were used (forward, reverse):
β2M, 5′-
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