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).