Quantitative microRNA expression analysis

The qPCR reaction was conducted with the miRcute microRNA qPCR Detection Kit (Tiangen, Beijing, China) on ABI PRISM 7500 Sequence Detection System (Applied Biosystems, Foster City, CA, USA). Each 20-μl qPCR reaction solution contained cDNA, 2× miRcute microRNA premix (with SYBR and ROX), the manufacturer-provided universal reverse primer, and a microRNA-specific forward primer (Tiangen, Beijing, China). The real-time PCR cycling conditions: 94°C for 2 min, 45 cycles at 94°C for 20 s, annealing at 60°C for 34 s, and extension at 72°C for 30 s. At the end of the real-time PCR reaction, a melting curve analysis was accomplished to ensure specific amplification of the expected PCR product.
The relative expression of the microRNAs was calculated from the equation log₁₀ (2^−ΔCt) with cel-miR-39. The ΔCT was calculated by subtracting the CT values of the cel-miR-39 from those of the microRNAs of interest [23 (link)].