Mirna rt pcr kit

Female BALB/c mice (6~8 weeks old, 20 ± 2 g) were obtained from the Laboratory Animal Centre of Guangdong Medical College. All mice with access to chow and water ad libitum were acclimated to laboratory conditions (50±10% humidity and 14/10 h light/dark cycles at 22±1°C) for at least one week before use in experiments. The mice were euthanized by cervical dislocation after intraperitoneal administration of 1% pentobarbital sodium (50 mg/kg, Sigma, P3761) after the experimental endpoint. Mice did not become severely ill or moribund during the experiments. All procedures involving animals were approved by the Laboratory Animal Centre of Guangdong Medical College Animal Care and Use Committee and conform to the Guide for Care and Use of Laboratory Animals.
Anti-β-catenin polyclonal antibody (cell signaling 8480), anti-β-actin antibody (cell signaling 4967S), HRP-conjugated anti-mouse IgG (CST, 7076), and HRP-conjugated anti-rabbit IgG (CST, 7074) were used for western blot. Kits used included miRNA RT-PCR Kit (Takara, RR716), miRcute miRNA Kit (Tiangen, DP501), and Dual-Luciferase Reporter Assay System (Promega, E1910). Lipofectamine 3000 (Invitrogen, L3000015) was used for transfection experiments. All oligonucleotide including miRNA mimic, miRNA inhibitor and their controls were chemically synthesized by Biomics Biotechnologies Co., Ltd. (Suzhou, China).

Real-time quantitative PCR (RT-qPCR) was conducted as previously described [5 ]. Briefly, total RNA containing small RNAs (microRNAs) was extracted from cells using mirVana™ Protein and RNA Isolation System™ Kit (Applied Biosystems, Ambion Inc) or TRI reagent (Sigma) according to the manufacturer's instructions. Total RNA and microRNA specific cDNA synthesis was performed with Prime Script RT Master Mix Perfect Real Time kit (DRR036A, Takara, China) and miRNA RT-PCR Kit (RR716, Takara, China), respectively. The resultant cDNA was diluted to a working concentration of 5ng/μl and stored at -20°C. The qRT-PCR reaction was run on ABI Prism 7500 system, using NCode™ EXPRESS SYBR® GreenER™ qPCR SuperMix Universal for miRNA RT-qPCR or Takara premix Ex Taq II (DRR820A, Takara, China) for others. Sequence for each primer was shown in Supplementary Table 1. Expression level of relative mRNA or microRNA was defined as the ratio of target gene expression level or microRNA expression level to 18S or U6 snRNA expression level, respectively, with that of the control sample set as 1.0.