Reverse transcription-quantitative (RT-q) PCR

Total RNA from SUM159 cells transfected with miR-203 mimics or miR-203 NC was extracted using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific Inc.) and cDNA was synthesized using iScript™ cDNA Synthesis Kit (Biorad Laboratories Inc.) following the manufacturer's instructions. iQ™ SYBR Green supermix (Biorad Laboratories Inc.) was used to perform RT-qPCR. A 7500 HT Fast Real-Time PCR system (Applied Biosystems; Thermo Fisher Scientific Inc.) was used to cycle and quantify reactions. The thermocycling conditions were as follows: denaturation at 95°C for 20 sec, followed by 40 cycles of 95°C for 10 sec, 60°C for 20 sec and 70°C for 10 sec. Relative gene expression levels of miR-203 were evaluated. U6 was used as the miRNA endogenous normalization control. The relative expression levels of FKBP5 were evaluated relative to glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The primer sequences used were as follows: miR-203 forward 5′-GGGGTGAAATGTTTAGGAC-3; reverse 5′-CAGTGCGTGTCGTGGAGT-3′; U6 forward 5′-CTCGCTTCGGCAGCACA-3′, reverse 5′-AACGCTTCACGAATTTGCGT-3′; FKBP5 forward 5′-GGGGTGAAATGTTTAGGAC-3; reverse 5′-CAGTGCGTGTCGTGGAGT-3′; and GAPDH forward 5′-TCAAGAAGGTGGTGAAGCAG-3′, reverse 5′-CGCTGTTGAAGTCAGAGGAG-3′. The relative expression of each target amplicon was calculated using the 2^−ΔΔCq method (12).