C2C12 culture: myogenesis timecourse and shRp58 treatment

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 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.

为预测参与肌生成的Rp58调控基因，本研究开展了RNA表达谱分析（RNA profiling）实验，对比了表达与不表达Rp58短发夹RNA（short hairpin RNA, shRNA）的C2C12细胞的RNA样本。结果显示，相较于对照C2C12细胞，稳定表达shRNA-Rp58的C2C12细胞中共有271个基因上调。鉴于Rp58在C2C12细胞中为转录抑制因子，我们提出假设：Rp58调控的基因簇其表达水平会随Rp58表达及肌生成进程而下调。据此，我们还通过微阵列分析，对C2C12肌生成实验的4个不同阶段（生长培养基GM、第0天、第2天、第4天）的基因动态表达模式进行了表征，共鉴定出399个在肌生成过程中呈下调趋势的基因。值得注意的是，该下调基因集与shRNA介导Rp58沉默后上调的基因存在高度重叠。
关键词：时间序列
C2C12小鼠骨骼肌细胞购自美国典型培养物保藏中心（American Type Culture Collection, ATCC）。细胞于生长培养基（GM，即添加10%胎牛血清FBS的达尔伯克改良伊格尔培养基DMEM）中培养。细胞在GM中增殖至汇合后，将培养基更换为分化培养基（DM，即添加2%马血清的DMEM）并继续孵育，每2天更换一次培养基。本实验均使用传代5次以内的细胞进行培养。
针对Rp58的shRNA实验采用Lipofectamine 2000（Invitrogen）进行转染。通过为期两周的筛选获得稳定转染的细胞株。
微阵列分析：按照前述方法从C2C12细胞中提取总RNA，合成互补RNA（cRNA）。取10 μg cRNA与Affymetrix小鼠基因组430 2.0芯片进行杂交。采用RMA算法对信号强度值进行定量分析。使用MAPPFinder（www.genmapp.org）将表达数据与已知通路进行整合分析。