RNA profiling of skeletal muscle in RP58 knockout mice

To predict RP58-regulated genes involved in skeletal myogenesis, RNA profiling experiments were performed, comparing RNA derived from skeletal muscle tissue of a RP58+/+ mouse to that from a RP58 knockout (KO) mouse at E18.5. Importantly, well-known dominant-negative inhibitors of muscle differentiation, the Id family of genes (Id1/Id2/Id3), were upregulated in the RP58 KO muscle. On the contrary, a number of muscle differentiation-related genes, such as Ckm, troponin and Myosin, were downregulated in the same sample. These results indicate that the repressor protein RP58 is important for muscle terminal differentiation, possibly suppressing the gene expression of muscle differentiation genes such as the Ids. Keywords: Knockout tissue Skeletal muscles of WT and RP58 KO mice (E18.5 diaphragm) were dissected under a microscope and RNA was extracted using ISOGEN (Nippongene). After EtOH precipitation, RNA was dissolved into DEPC-DW and its concentration was determined. Microarray analysis - cRNA was synthesized. 10 ug of cRNA were hybridized to Affymetrix mouse 430A 2.0 arrays. Signal intensities were calculated using the RMA method. MAPPFinder (www.genmapp.org) was used to integrate expression data with known pathways.为预测参与骨骼肌发生的RP58调控基因，本研究开展RNA表达谱分析实验，对比了胚胎发育第18.5天（E18.5）RP58野生型（RP58+/+）小鼠与RP58基因敲除（knockout, KO）小鼠的骨骼肌组织来源RNA。值得注意的是，作为已知的肌肉分化显性负调控因子，Id家族基因（Id1/Id2/Id3）在RP58 KO小鼠的肌肉组织中表达上调；与之相反，包括肌酸激酶（Ckm）、肌钙蛋白及肌球蛋白在内的多种肌肉分化相关基因在该样本中表达下调。上述结果表明，阻遏蛋白RP58对肌肉终末分化具有关键作用，其可能通过抑制Id家族等肌肉分化基因的转录表达来发挥调控功能。 关键词：敲除组织 显微解剖获取野生型（wild type, WT）与RP58 KO小鼠E18.5膈肌骨骼肌组织，采用ISOGEN（Nippongene公司）提取总RNA。经乙醇沉淀后，将RNA溶解于DEPC处理去离子水（DEPC-DW）中，并测定其浓度。 芯片微阵列分析：合成互补RNA（cRNA），取10 μg cRNA与Affymetrix小鼠430A 2.0芯片进行杂交。采用RMA算法计算信号强度。使用MAPPFinder（www.genmapp.org）整合表达数据与已知通路信息。