Quantifying miR-328-3p Expression in Tissues

qRT-PCR was performed to detect the expression levels of miR-328-3p in human tissues and cell lines according to our previous study.16 (link) Total RNA was extracted from fresh tissues and cells using TRIzol reagent (Thermo Fisher Scientific). TaqMan miR Assays (Thermo Fisher Scientific) with primers specific to miR-328-3p were used. Reverse transcription was performed using One Step PrimeScript miR cDNA Synthesis Kit (Thermo Fisher Scientific), and qRT-PCR was performed using SYBR Premix Ex Taq™ II (Thermo Fisher Scientific). RNU6B was used as an internal control. qRT-PCR primer sequences were as follows: miR-328-3p, forward 5′-CTG GCC CTC TCT GCC C-3′, and reverse 5′-GTG CAG GGT CCG AGG T-3′; RNU6B, forward 5′-ACA GUA GUC UGC ACA UUG GUU A-3′, and reverse 5′-ACG CAA ATT CGT GAA GCG TT-3′. Quantitative PCR was performed using an ABI 7500 Real-Time PCR Detection System (Thermo Fisher Scientific). The threshold cycle (Ct) was defined as the fractional cycle number at which the fluorescence passed the fixed threshold. Each sample was detected thrice, and the relative expression level of miR-328-3p to RNU6 was calculated using the equation 2^−ΔCt, where ΔCT = (CT_miR-328-3p/CT_RNU6). The median value of miR-328-3p expression in osteosarcoma tissues was used as a cutoff point for dividing miR-328-3p-low/high groups.