Mirna sybr qrt pcr kit

cDNA was synthesized from miRNA by using miRNA cDNA First Strand Synthesis or from mRNA by using PrimeScript RT reagent Kit (Takara, Ohtsu, Japan). Quantitative realtime PCR (qRT-PCR) for pre-miR-146a was performed with SYBR Premix Ex Taq II (TaKaRa) with a BIO-RAD Multicolour Real-time PCR Detection System (iQTM5). The primers we used are listed here: pre-miR-146a (forward 5′-TGAGAACTGAATTCCATGGGTT-3′ and reverse 5′-CTGAAGAACTGAATTTCAGAGG-3′), β-actin (forward 5′-AGAAAATCTGGCACCACACC-3′and reverse 5′-AGAGGCGTACAGGGATAGCA-3′). The cycling conditions are as follows: 95°C for 2 min., followed by 45 cycles of denaturation at 95°C for 5 sec., annealing at 57°C for 10 sec. and extension at 72°C for 15 sec. QRT-PCR for miR-146a was performed with miRNA SYBR qRT-PCR Kit (Qiagen, Valencia, CA, USA) with iQTM5. The primers of RUNU6, and hsa-miR-146a were designed and synthesized by Tiangen Company based on miRBase (http://www.mirbase.org). DNA was amplified for 40 cycles of denaturation for 20 sec. at 95°C and annealing for 60 sec. at 60°C. All reactions were run in triplicates for at least three independent experiments. Relative quantification was performed according to the ΔΔCT method, and results were expressed in the linear form by using the formula 2^−ΔΔCT24 (link).