C2C12 culture: myogenesis timecourse and shRp58 treatment
ABSTRACT: To predict Rp58-regulated gene involved in myogenesis, RNA profiling experiments were performed, comparing RNA derived from C2C12 with or without expressing shRNA for Rp58. As a result, 271 genes were upregulated in C2C12 stably expressing shRNA-Rp58 cells compared with control C2C12 cells. As Rp58 is repressor in C2C12, we hypothesized that Rp58 regulates gene cluster which expression is downregulated in accordance with Rp58 expression and myogenesis progression. In this regard, we also characterized dynamic gene expression patterns during myogenesis by microarray at 4 different stage (GM, day 0, 2, 4) of C2C12 myogenesis assays and found that 399 genes expression is characterized as downregulation pattern during myogenesis. Importantly, this down regulation gene set and upregulated genes by shRNA for Rp58 were highly overlapped. Keywords: time course, shRNA Overall design: C2C12 murine skeletal muscle cells were purchased from American Type culture Collection (ATCC). These cells were mainteined in GM (DMEM supplemented with 10% FBS). Cells were grown in GM and after reaching counfluence, the medium was switched to DM (DMEM supplemented with 2% hourse serum) and further incubated. The medium was changed every 2 days. Culture was performed by using within five passages cells. For the experiment of shRNA for Rp58, transfection was performed by using Lipofectamin 2000 (Invitrogen). Stable transfectants were obtained by selection of the transfected C2C12 cells for two weeks. Microarray analysis - RNA was isolated as described from C2C12, and cRNA was synthesized. 10 ug of cRNA were hybridized to Affymetrix mouse 430 2.0 arrays. Intensity values were quantified using RMA algorithm. MAPPFinder (www.genmapp.org) was used to integrate expression data with known pathways.