Mir x mirna q rt pcr kit

Total RNA was extracted from cells by using RNAiso Plus (TaKaRa, Dalian, China) following the manufacturer's instructions. RNA was then reverse transcribed to cDNA with Mir-X miRNA First-Strand Synthesis kit (TaKaRa). Real-time PCR was carried out using Mir-X miRNA q-RT PCR kit (TaKaRa). The real-time PCR reaction contained: 9 µL of ddH₂O, 12.5 µL of SYBR Advantage Premix (2X), 0.5 µL of ROX Dye (50X), 0.5 µL of miR21-specific primer (10 µM), 0.5 µL of mRQ 3' primer (10 µM), 2.0 µL of cDNA template. The program was 95°C for 10 s, followed by 40 cycles of 95°C for 5 s, 60°C for 20 s. The relative expression level of miR21 was normalized to that of internal control U6 using the comparative threshold cycle (Ct) method (ΔΔCt method). The miR21-specific primer sequence was 5'-TAGCTTATCAGACTGATGTTGA-3'. The mRQ 3' primer, U6 Forward primer and U6 Reverse primer were provided by q-RT PCR kit (TaKaRa).

The cDNA was synthesized using Mir-X miRNA First-Strand Synthesis kit (Takara, Dalian, China) according to the manufacturerʼs protocols. The expression levels of miRNA were measured using Mir-X miRNA qRT-PCR Kit (Takara, Dalian, China) on an ABI 7900 real-time PCR system (Applied Biosystems, CA). Exogenous cel-miR-39–3p was used as a control. The relative expression levels of miRNAs in plasma were calculated using the equation 2^−ΔΔCt.