2.3. Real-time quantitative PCR (RT-qPCR)

RT-qPCR was performed as described before [¹⁰]. Our crew extracted total RNA taking advantage of TRIzol reagent (Life Technologies Corporation) as well as RNeasy Mini kit (Qiagen, Duesseldorf, Germany), and then purified. For measuring and analyzing miR-183-5p, our team reverse-transcribed RNA exploiting TaqMan® MicroRNA Reverse Transcription Kit (Thermo Fisher Scientific). Besides, we used TaqMan® Universal Master Mix II (Applied Biosystems) for assisting TaqMan® MicroRNA analysis to carry out RT-qPCR. To determine and analyze PDCD4, we reverse-transcribed RNA samples applying Multiscribem reverse transcription kit (Applied Biosystems), and we completed RT-qPCR utilizing Fast START Universal SYBR Green Master (ROX) (Roche). We used the 2^−ΔΔCt method to analyze data. MiR-183-5p and PDCD4 mRNA expression were normalized to U6 and β-actin, respectively. The primer sequences were: miR-183-5p, 5ʹ-TATGGCACTGGTAGAATTCACT-3ʹ (Forward) and 5ʹ-ACGCTTCACGAATTTGCGT-3ʹ (Reverse); PDCD4, 5ʹ-AGAGACAGAAGAGCGGGGT-3ʹ (Forward) and 5ʹ-CCAGCATTTTCTGCAGGGTTT- 3ʹ (Reverse); U6, 5ʹ-TGCGGGTGCTCGCTTCGGCAGC-3ʹ (Forward) and 5ʹ-CCAGTGCAGGGTCCGAGGT-3ʹ (Reverse); β-actin, 5ʹ-CCTTCCTGGGCATGGAGTCCT-3ʹ (Forward) and 5ʹ-GGAGCAATGATCTTGATCTT- 3ʹ (Reverse).