Cdnas

RNAs were isolated from blood samples of NSCLC patients using the commercial RNA isolation kit (TIANGEN Biotech, Beijing, China) and reversely transcribed to cDNAs (Applied Biosystems, Foster City, CA, USA). Amplification conditions were as follows: 95°C at 5 min and 35 cycles at 95°C for 10 s, 60°C for 30 s, and 72°C for 30 s. Primer sequences were as follows: AK021443, F: 5′-CTTGAACCCAGAAGACAGG-3′, R: 5′-ATGGAACATTAGAGGTAGCAC-3′; GAPDH, F: 5′-CGGATTTGGTCGTATTGGG-3′, R: 5′-GATTTTGGAGGGATCTCGC-3′.

Total RNAs were extracted using Trizol reagents (Thermo, USA) according to the manufacturer’s protocol. The quantity and quality of total RNA samples were determined by a NanoDrop machine (DE, USA). Then, 0.5 μg of RNA extracted from clinical tissues or cells was subjected to reverse transcription and cDNAs were obtained (Applied Biosystems, USA) according to the manufacturer’s instructions. To detect the expression of miR-328-3p in the OS tissues and OS cell lines, qRT-PCR analysis was conducted using SYBR Green qPCR Master Mix (Roche, Switzerland). The reaction was conducted by using 1 μL cDNA, 10 μL SYBR Green qPCR Master Mix, 2 μL primers and 7 μL ddH₂O, with a total volume of 20 μL. The conditions were as follows: 95 °C for 30 s, followed by 40 cycles of amplification at 95 °C for 5 s, at 59 °C for 30 s and at 72 °C for 30 s. The primer sequences used in our study were as follows: miR-328-3p forward, 5′-CGGGCCTGGCCCTCTCTGCC-3′ and reverse, 5′-CAGCCACAAAAGAGCACAAT-3′; U6 forward, 5′-CTCGCTTCGGCAGCACA-3′ and reverse, 5′-AACGCTTCACGAATTTGCGT-3′. The expression of miR-328-3p was normalized to that of U6 small nuclear RNA. The expression of miR-328-3p was quantified using the 2^−ΔΔCT method. ΔC_T = C_T (miR-328-3p)-C_T (U6). C_T was threshold cycle number.