Mircute microrna qpcr detection kit

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)].

Total RNA, including mRNA and microRNA (miRNA), was extracted from cells and tissue using the miRcute miRNA Isolation Kit (Tiangen, Shanghai, China) according to the manufacturer’s manual. Reverse-transcription was performed with FastKing gDNA Dispelling RT SuperMix (Tiangen). Quantitative real-time PCR (qPCR) analysis of pri-miR-105, and GR levels were performed with a SuperReal PreMix Plus (SYBR Green) using 40 cycles of amplification (95 °C for 10 s, 60 °C for 25 s, and 72 °C for 20 s). MiR-105 was reverse-transcribed using a microRNA first-strand cDNA synthesis kit from Tiangen. qPCR was performed on a Mastercycler ep realplex Real-time PCR System of eppendorf (40 cycles of amplification: 94 °C for 2 min, 94 °C for 20 s, and 60 °C for 34 s) using a miRcute microRNA qPCR detection kit (Tiangen). The primers for miR-105, pri-miR-105, and U6 were synthesized by Tiangen. Those for GR (RQP048912), and Gapdh (RQP049537) were purchased from GeneCopoeia (Guangzhou, China). To obtain the fold-change in mRNA and miRNA levels, the data were analyzed using the 2^–ΔΔCT method. The final gene expression levels were normalized to that of Gapdh for mRNA and U6 for miRNA. Triplicate reactions were carried out in three separate experiments.