Taqman microrna assay kit

Total RNA was extracted from cultured cells and fresh tissues using TRIzol reagent (Invitrogen). Total miRNAs were extracted using a mirVana miRNA Isolation Kit (Ambion) from paraffin-embedded OS specimens according to the manufacturer’s protocol. The levels of the miRNAs and the RNU6 endogenous control were measured using TaqMan primer sets and a TaqMan microRNA Assay Kit (Life Technologies). For the expression analysis of other genes, reverse transcription (RT) and PCR were performed with a High-Capacity cDNA Reverse Transcription kit and a QuantiTect SYBR Green PCR kit (QIAGEN), respectively. The primer sequences are listed in Table S2.

Fetal bovine serum (FBS), puromycin, 4-(Methylnitrosoamino)-1-(3-pyridinyl)-1-butanone, and cell culture medium were purchased from Sigma (St. Louis, MO, USA). Antibodies against total Akt, Akt1, Akt2, DNMT1, phosphor-Akt (ser473), phosphor-mTOR (ser2448), phosphor-p70S6K (Thr 389), Ki-67, and actin were obtained from Cell Signaling Technology, Inc. (Danvers, MA, USA). Negative control agomiR and miR-124 agomiR were synthesized at Integrated DNA Technologies (Coralville, IA, USA). MK2206 was purchased from Selleckchem (Houston, TX, USA). Plasmid of constitutively active Akt and mutant K-ras (G12D) were kindly gifted from Dr. Cheng (Moffitt Cancer Center). The expression vector of miR-124 and miR-124-antisense were from GeneCopoeia (Rockville, MD, USA). Dual-Luciferase Assay Kit and TUNEL assay kit were purchased from Promega (Madison, WI, USA). SYBR Green PCR kit, Lipofectamine 2000, Taqman MicroRNA Assay kit, TRIzol, Opti MEM, High-Capacity cDNA Reverse Transcription kit, miRNA expression reporter vector, miR-124 mimics, miR-124 and RNU6 primer sets, and antisense of miR-124 were purchased from Life Technologies (Carlsbad, CA, USA). siRNA for target DNMT1 (#1, 5′-CGAGUCUGGUUUGAGAGUTT-3′; #2, 5′-GGAAUGGCAGAUGCCAACAGCTT-3′) was generated by RiboBio (Guangzhou, China).