Total RNA was extracted from cells and tissues using TRIzol® reagent (Invitrogen; Thermo Fisher Scientific, Inc.). Total RNA was reverse transcribed into cDNA using the Primer-Script™ One Step RT-PCR kit (Takara Biotechnology Co., Ltd., Dalian, China). qPCR was subsequently performed using SYBR® Premix Ex Taq (Takara Biotechnology Co., Ltd.), according to the manufacturer's protocol. The following thermocycling conditions were used: 95°C for 5 min; 40 cycles of 95°C for 10 sec, 60°C for 20 sec and 72°C for 20 sec. Differences in the expression of Bcl-2-associated X (Bax), B-cell lymphoma-2 (Bcl-2), caspase-3 and mammalian target of rapamycin (mTOR) in the four groups were compared. The relative mRNA expression levels were quantified using the 2−ΔΔCq method (26 (link)) and normalized to the internal control GAPDH.
For the detection of miRNA, total RNA was isolated using the miRNA Extraction kit (Qiagen GmbH, Hilden, Germany). Total RNA was reverse transcribed into cDNA using the miRNA cDNA Synthesis kit (Qiagen GmbH). qPCR was subsequently performed using the SYBR® Green (Takara Biotechnology Co., Ltd.), according to the manufacturer's protocol. The experiment was performed as described above. U6 served as an internal control. The sequences of the primers utilized are listed inTable I .
For the detection of miRNA, total RNA was isolated using the miRNA Extraction kit (Qiagen GmbH, Hilden, Germany). Total RNA was reverse transcribed into cDNA using the miRNA cDNA Synthesis kit (Qiagen GmbH). qPCR was subsequently performed using the SYBR® Green (Takara Biotechnology Co., Ltd.), according to the manufacturer's protocol. The experiment was performed as described above. U6 served as an internal control. The sequences of the primers utilized are listed in