Quantitative PCR analysis of microRNAs

The PCR reaction was performed for amplification using the miRcute microRNA qPCR Detection Kit (Tiangen, Beijing, China) on ABI PRISM 7500 Sequence Detection System (Applied Biosystems, Foster City, CA, USA). Each qPCR reaction solution contained diluted cDNA, 2× miRcute microRNA premix (with SYBR and ROX), the manufacturer-provided microRNA-specific forward primer, and a universal reverse primer to a total volume of 20 μl. The qPCR reaction parameters were 94 °C pre-denaturation for 2 min, 45 cycles of 94 °C for 20 s, 60 °C annealing for 34 s, and 72 °C extension for 30 s. A melting curve analysis was accomplished to ensure the specificity of the target PCR product in the end.
The relative expression of the microRNAs was calculated using the equation log₁₀ (2^−ΔCT). The ΔCT was equal to CT values of the microRNAs of interest minus the CT values of the cel-miR-39 [19 (link)].