Quant sybr green pcr kit

Antibodies against phosphorylated α (p-AMPKα Thr172) and non-phosphorylated AMPKα were purchased from Cell Signaling Technology (Danvers, MA, USA); Antibody against Bmi-1 was from Abcam (Cambridge, MA, USA); Antibodies against LKB1 and β-actin were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-LITAF antibody was from Abnova Corporation (Taiwan, China). Diaminobenzidine (DAB) was from Dako (Carpinteria, CA, USA). Enzyme Antibody Conjugates were from ZSGB-BIO (Beijing, China). Metformin and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were from Sigma-Aldrich (St Louise, MO, USA). Trizol reagent, FastQuant RT Kit, and Quant SYBR Green PCR kit were from TIANGEN Biotech (Beijing, China). Lipfectamine 2000 was from Life Technologies (Grand Island, NY). miR-15a mimic, miR-128 mimic, miR-192 mimic, miR-194 mimic, and negative control mimic were purchased from Ribobio (Guangzhou, China). SuperSignal West Pico Chemiluminescence kit was from Thermo-Fisher scientific (Waltham, MA, USA).

Fresh frozen tumor tissue samples (n = 24) and matched adjacent samples were collected for quantitative real-time polymerase chain reaction (qRT-PCR) analysis to investigate the difference of KIF2A expression between cancerous and normal tissues. Total tissue ribonucleic acid (RNA) was extracted using the RNeasy Mini Kit (Qiagen, Valencia, CA, USA). qRT-PCR analysis was performed according to the manufacturer's instructions (Quant SYBR Green PCR Kit, Tiangen Biotech, Beijing, China). The KIF2A primer sequences were as follows: forward 5′-GCCTTTGATGACTCAGCTCC-3′ and reverse 5′-TTCCTGAAAAGTCACCACCC-3′. Moreover, the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene as the housekeeper gene was run in parallel to normalize KIF2A gene expression (forward primer 5′-TGCACCACCAACTGCTTAGC-3′ and reverse primer 3′-GGCATGGACTGTGGTCATGAG-5′). For relative quantification, 2^−ΔΔCt was calculated and used as an indicator of the level of enzyme expression. All analyses of the samples were performed in triplicate.

Total RNA was extracted using the RNeasy Mini Kit (Qiagen, Valencia, CA). Quantitative Real-time PCR was performed according to the manufacturer’s instructions (the Quant SYBR Green PCR Kit, Tiangen Biotech, Beijing). ACTB (beta-actin) and HPRT1 (hypoxanthine phosphoribosyltransferase 1) were used as loading controls for cell lines and tissues, respectively. The conditions were as follows: 10 min at 95°C, followed by 40 cycles of 95°C for 15 sec and 60°C for 60 sec. The primers were shown at Supplementary Table S2. ACTB and HPRT1 were used as loading control. The relative quantification was calculated by 2^−ΔCt method (ΔCt = Ct^[target] − Ct^[HKG]) for cell lines or 2^−ΔΔCt method [28] (link) (−ΔΔCt = ΔCt^[normal] −ΔCt^[tumor]) for tissues.