miRNA sequencing of rat hearts treated chronically with rofecoxib or its vehicle
miRNA sequencing of rat hearts treated chronically with rofecoxib or its vehicle
The hearts of the animals were removed and subjected to Langendorff perfusion for 2 minutes at 37°C. During this perfusion, the hearts were flushed with oxygenated Krebs-Henseleit solution (in millimolar (mM): NaCl 118, KCl 4.7, MgSO4 1.2, CaCl2 1.25, KH2PO4 1.2, NaHCO3 25, and glucose 11) to eliminate any remaining blood from the tissue. Afterwards, the samples were snap-frozen in liquid nitrogen and stored at -80 °C.  For in vivo experiments, two-month-old male Wistar rats of 189–287 g were randomised and treated daily for 28 ± 1 days by oral gavage with 5.12 mg kg−1 rofecoxib or with its vehicle (1% hydroxyethylcellulose).  The total RNA of the left ventricle tissue samples (n = 8/group) was extracted by employing the RNAzol RT kit (Sigma Aldrich, St. Louis, MO, USA) according to the manufacturer´s instructions. RNA concentrations of all samples were quantified using spectrophotometry (NanoDrop; Thermo Fischer Scientific, Waltham, MA, USA) and RNA HS Assay Kit with Qubit 3.0 Fluorometer (Thermo Fisher Scientific, Waltham, MA, USA). The RNA Integrity Numbers were determined by 2100 Bioanalyzer Instrument (Agilent Technologies, Santa Clara, CA, USA) and agarose gel electrophoresis. We employed the NEBNext Multiplex Small RNA Library Prep Set for Illumina (New England Biolabs, Ipswich, MA, USA) for small RNA library construction according to the manufacturer's protocol. Libraries were quantified and qualified using High Sensitivity DNA1000 ScreenTape system with 2200 Tapestation (Agilent Technologies, Santa Clara, CA, USA) and dsDNA HS Assay Kit with Qubit 3.0 Fluorometer (Thermo Fisher Scientific, Waltham, MA, USA).