Six1 homeoprotein drives myofiber type IIA specialization in soleus muscle

Six homeoproteins regulate fast MYH expression and calcium homeostasis soleus muscles were collected from cSix1 KO and control mice. Total RNAs were extracted by Trizol Reagent (Invitrogen) according to manufacturer's instruction. After validation of RNA quality with the Bioanalyzer 2100 (using Agilent RNA6000 nano chip kit), 50 ng of total RNA were reverse transcribed following the Ovation PicoSL WTA System (Nugen). Briefly, the resulting double-strand cDNA was used for amplification based on SPIA technology. After purification according to Nugen protocol, 5 ÎŒg of single strand DNA was used for generation of Sens Target DNA using Ovation Exon Module kit (Nugen). 2.5 ug of Sens Target DNA were fragmented and labelled with biotin using Encore Biotin Module kit (Nugen). After control of fragmentation using Bioanalyzer 2100, the cDNA was then hybridized to GeneChip® Mouse Gene 1.0 ST (Affymetrix) at 45°C for 17 hours. After overnight hybridization, the ChIPs were washed using the fluidic station FS450 following specific protocols (Affymetrix) and scanned using the GCS3000 7G. The scanned images were then analyzed with Expression Console software (Affymetrix) to obtain raw data (cel files) and metrics for Quality Controls. The analysis of some of these metrics and the study of the distribution of raw data show no outlier experiment.本研究采集cSix1敲除（cSix1 KO）小鼠与对照小鼠的比目鱼肌，以探究六种同源框蛋白（homeoproteins）对快肌MYH（fast MYH）表达及钙稳态的调控作用。按照制造商操作说明，采用Trizol试剂（Invitrogen）提取总RNA。使用生物分析仪2100（配套安捷伦RNA6000纳米芯片试剂盒）对RNA质量进行验证后，取50 ng总RNA，按照Ovation PicoSL WTA系统（Nugen）的操作流程进行逆转录反应。简言之，所得双链cDNA将基于SPIA技术进行扩增。按照Nugen试剂盒说明书完成纯化后，取5 μg单链DNA，使用Ovation外显子组模块试剂盒（Nugen）制备靶向传感DNA（Sens Target DNA）。取2.5 μg靶向传感DNA，采用Encore生物素标记模块试剂盒（Nugen）进行片段化与生物素标记。使用生物分析仪2100验证片段化效果后，将cDNA与GeneChip®小鼠基因1.0 ST芯片（Affymetrix）在45℃下杂交17小时。过夜杂交完成后，按照Affymetrix的专用操作流程，使用流体工作站FS450对ChIPs进行洗涤，并通过GCS3000 7G扫描仪完成扫描。随后使用Expression Console软件（Affymetrix）对扫描得到的图像进行分析，以获取原始数据（cel格式文件）与质量控制指标。对部分质量控制指标及原始数据分布的分析结果显示，本实验无异常样本。