RNA extraction and quantification Total RNA was extracted working with Trizol based on the manufac turers directions. Reverse transcription was performed employing One particular Step PrimeScriptmiRNA cDNA Synthesis Kit. True time PCR was performed applying SYBRPremix Ex TaqTM II with an iCyclerthermal cycler. U6 RNA was applied as a miRNA internal control. The primers of miR 92b was as follows, Colony formation assay U251 and U87 cells had been transfected with miR 92b mimics, a manage oligonucleotide and a miR 92b inhibitor. Immediately after the transfection, the U251 and U87 cells have been counted and seeded in 12 well plates at a density of 50 and 60 cells per properly, respectively. The culture medium was replaced just about every 3 days. The number of colonies was counted around the sixth day just after seeding.
The price of colony formation extra resources was calculated using the following equation, colony formation price ? 100%. MTT assays The MTT assay was employed to decide cell viability. Each of the cells had been seeded into 96 properly culture plates in standard development medium. The cells transfected with miR 92b mimics. control and inhibitors have been grown for four days. One particular plate was developed immediately soon after the medium transform as well as other plates were developed each and every 24 hours for four days. Assays were initiated by adding 20 L of MTT substrate to every well and incubating the cells for an further three hours. Ultimately, the medium was removed and 200 L DMSO was added to every nicely. The absorbance was measured at 492 nm applying an Automated Microplate Reader. RT PCR Analysis was employed to establish the relative expression levels of miRNAs.
Total RNA was isolated making use of TRI ZOLTM reagent according to the suppliers instruction. Reverse transcrip tion was carried out utilizing One Step PrimeScript miRNA cDNA Synthesis Kit. Genuine time PCR was performed working with SYBR Green Supermix with an iCyclerthermal cycler. Primers of all genes have been in Supple mentary. The find more information data had been collected and analyzed applying the comparative Ct system using GADPH as the reference gene. MicroRNA target prediction The target genes of miR 92b were predicted by the fol lowing computer aided algorithms, TargetScan Human Release six. two. Luciferase assay The three UTR of human DKK3, containing the putative target web sites for miR 92b, was amplified by PCR. The wild type and mutant inserts were transfected in to the PGL3 promoter vector. Dual Luciferase reporter assays were performed according to the companies directions as previously described.
Flow cytometric analysis of apoptosis Cells were plated in six properly plates in antibiotic totally free medium and transfected with manage oligonucleotide or inhibitor working with Lipofectamine 2000 based on the makers recommendation. Luciferase and renilla signals were measured 48 h immediately after transfection using the Annexin V FITC apoptosis detection kit as described by the manufac turers directions.