Effect of glucocorticoid steroid regimens on murine dystrophic muscle. Mus musculus

The goal of this study is to compare the effect of weekly and daily steroid regimens (prednisone) on transcriptional regulation of candidate genes for muscle repair and atrophic response in mdx mice (n=5 mice/group). Overall design: Total RNA was isolated from ~30mg muscle tissue (quadriceps muscles from treated and control DBA/2J-mdx male 6 month-old mice) with the RNeasy Protect Mini Kit as per manufacturer’s instructions. RNA was quantitated at the Qubit fluorometer and quality-controlled at a 2100 Bioanalyzer. Libraries were prepared from approximately 1ug RNA/sample by means of TruSeq Stranded Total RNA Library Prep Kit. Libraries were sequenced through the Illumina NextSeq 500 System (high-throughput, paired-end 150bp fragment sequencing. Reads from each sample were aligned with TopHat v2.1.0 to the mm10 genome assembly (grcm38, version 78). Transcripts were assessed and raw read counts per gene were quantified with HTseq. Reads Per Kilobase of transcript per Million mapped reads (RPKM) and fold-changes between groups were calculated using EdgeR from the Bioconductor package. Differentially expressed genes were identified by adjusted P-value <0.05.

本研究旨在比较每周与每日糖皮质激素方案（泼尼松，prednisone）对mdx小鼠（每组n=5只）肌肉修复候选基因的转录调控及萎缩反应的影响。
总体实验设计：从经处理及对照的6月龄雄性DBA/2J-mdx小鼠的股四头肌中取约30mg肌肉组织，依照制造商说明书使用RNeasy Protect Mini试剂盒（RNeasy Protect Mini Kit）提取总RNA。通过Qubit荧光计（Qubit fluorometer）对RNA进行定量，并利用2100生物分析仪（2100 Bioanalyzer）完成质量控制。以每份样本约1μg RNA为起始材料，通过TruSeq链特异性总RNA文库制备试剂盒（TruSeq Stranded Total RNA Library Prep Kit）构建测序文库。采用Illumina NextSeq 500测序系统（Illumina NextSeq 500 System，高通量双端150bp片段测序）对文库进行测序。将每个样本的测序读数通过TopHat v2.1.0比对至mm10基因组组装版本（grcm38，版本78）。利用HTseq对转录本进行评估，并统计每个基因的原始读数计数。采用Bioconductor软件包（Bioconductor package）中的EdgeR软件包，计算每百万映射读数每千碱基转录本的读数（RPKM）及组间差异倍数。以校正后P值<0.05作为差异表达基因的筛选标准。