Quantitative reverse-transcription polymerase chain reaction (qRT-PCR)
Total RNA was extracted from normal H9C2 cells, hypoxic H9C2 cells, NC, miR-210 mimic and miR-210 inhibitor group with TRIzol reagent (Invitrogen, Carlsbad, USA) following the manufacturer’s instructions. Briefly, single-stranded complementary DNA (cDNA) was synthesized from 2 μg of RNA via the RevertAid First Strand cDNA Synthesis Kit (Fermentas, Ontario, Canada). Subsequently, the generated miR-210 cDNA was amplified via qRT-PCR based on the TaqMan microRNA assay protocol (Roche LightCycler 480 II System). Briefly, 20 µL reactions were incubated in a 96-well optical plate at 94°C for 2 min and then subjected to 40 cycles of 94°C for 20 s and 60°C for 34 s. The relative expression of miR-210 was normalized to the internal control (U6) using the 2–∆∆Ct method. The primers for miR-210 and U6 were synthesized with the miScript Primer Assay kit (Qiagen, Dusseldorf, Germany).