Total RNA was extracted using the E.Z.N.A.® Total RNA kit I (Omega Bio-tek) according to the manufacturer's instructions. The quality of the RNA was measured using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Inc.). For lncRNA and AKT2 mRNA, cDNA was synthesized using a M-MLV First-Strand cDNA Synthesis kit (Invitrogen; Thermo Fisher Scientific, Inc.). RT-PCR was performed at 65°C (10 min), on ice (2 min), 40°C (30 min) and 70°C (10 min). The amplification program was 50°C (2 min), 95°C (2 min), followed by 40 cycles of 95°C (15 sec) and 60°C (30 sec). For microRNAs (miRNAs or miRs), RT-PCR and RT-qPCR were performed using the microRNA reverse transcription and RT-qPCR kit (GenePharma, Inc.). qPCR was carried on a Bio-Rad CFX Manager (Bio-Rad Laboratories, Inc.). The reverse transcription program was 25°C (30 min), 42°C (30 min) and 85°C (30 min). The amplification program was 95°C (3 min), followed by 40 cycles of 95°C (12 sec) and 62°C (40 sec). A relative amount of transcripts was normalized using the 2−ΔΔCq method with β-actin for large mRNAs and U6 for miRNAs. The specific primers used are listed in Table I.

Sequences of primes used in the present study.

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