Micrornas quantitation pcr kit

Total RNA was prepared using Trizol reagent (Invitrogen). Then total RNA (1 μg) was reverse transcribed using the PrimeScript™ RT Reagent Kit (Takara Bio, Shiga, Japan) following the manufacturer's instructions. The qRT-PCR was performed using SYBR® Premix Ex Taq™ (Takara) or MicroRNAs Quantitation PCR Kit (Sangon Biotech, Shanghai, China). PCR was run on Realtime PCR system (Applied Biosystems, Carlsbad, CA) under the following condition: initial denaturation at 95 °C for 5 min, 95 °C for 20 s, annealing at 56 °C for 30 s, extension at 72 °C for 1 min, 35 cycles, and final extension at 72 °C for 5 min. Fold changes in expression of each gene were calculated using the 2^−ΔΔC_t method. And the C_t values for the mRNA and miR-873-5p were normalized to β-actin and U6, respectively.

To assess miR-let-7b expression in CD133⁺ OCSCs, qRT-PCR analysis was performed with internal standards. Total RNA was isolated from the harvested cells and reverse transcribed into cDNA with the RevertAid Reverse Transcriptase (Thermo Fisher Scientific) according to the manufacturer’s instructions. qRT-PCR was performed using the MicroRNAs Quantitation PCR Kit (Sangon Biotech). The relative expression of miR-let-7b relative to non-treated cells was calculated by the ΔΔC_t method in triplicate experiment.

Total RNA was extracted using TRIzol (Sangon Biotech, B511311, Shanghai, China) and stored at −80°C. A NanoDrop 2000 UV‐Vis spectrophotometer (Thermo Scientific, USA) was used to determine the concentration and quality of total RNA. Then, an M‐MuLV First‐Strand cDNA Synthesis kit (Sangon Biotech, B532435, Shanghai, China) was used for reverse transcription of total RNA. An Applied Biosystems 7500 Sequence Detection System was used to determine the mRNA levels with 2×SG Fast qPCR Master Mix (Low Rox) kit (Sangon Biotech, B639272, Shanghai, China). Each sample was analysed in triplicate. The β‐actin gene served as a control, and the data were analysed using the 2^−(ΔΔCt) method. To detect mature miR‐21‐5p or pri‐miR‐21, a microRNA First‐Strand cDNA Synthesis kit (Sangon Biotech, B532453, Shanghai, China) was used to synthesize cDNA following the manufacturer's protocol. A MicroRNAs Quantitation PCR Kit (Sangon Biotech, B532461, Shanghai, China) was used to conduct qRT‐PCR, and an Applied Biosystems 7500 Sequence Detection System was used to detect the levels of miR‐21‐5p and pri‐miR‐21. Each sample was analysed in triplicate. U6 small nuclear RNA served as a control, and the data were analysed using the 2^−(ΔΔCt) method. The sequences of all primers are listed in Table 1.