FUNCTIONAL ANALYSIS OF CTCF DURING MAMMALIAN LIMB DEVELOPMENT_gene expression profiling

Limb buds were dissected from E10.75 mouse embryos and stored in RNAlater reagent (Qiagen), for genotyping. For each replicate, RNA was isolated from pools of six limb buds either of wild type or homozygous mutants, using RNeasy micro-kit (Qiagen). cRNA was synthesized according to the manufacturer's instructions (Ambion) and hybridized to the GeneChip Mouse Genome 430 2.0 Arrays (Affymetrix), which interrogates ca 39,000 transcripts. Three independent RNA extractions, cDNA synthesis and array hybridizations were performed. The expression arrays data were normalized and scaled to signal intensity of 100 using GCOS 1.2 software (Affymetrix). Expression levels were analyzed using GeneSpring software (Silicon Genetics, Redwood City, CA) and Matlab 2009 (Math Works, Inc., MA). To identify differentially expressed transcripts, pair-wise comparison analysis was performed with GCOS 1.2 software (Affymetrix). A 77% cutoff in consistency of change (at least seven out of nine comparisons were either increased or decreased) was applied. Only genes that satisfied the pair-wise comparison test and displayed a 1.5 fold change in expression were considered for further analysis.从E10.75阶段的小鼠胚胎中分离肢芽，置于RNAlater储存液（Qiagen）中用于基因分型。每份生物学重复均取6个野生型或纯合突变体的肢芽混合样本，使用RNeasy微量试剂盒（Qiagen）提取总RNA。按照Ambion公司的操作说明合成互补RNA（cRNA），并将其与GeneChip小鼠基因组430 2.0阵列（Affymetrix）进行杂交，该阵列可检测约39000个转录本。本实验共开展3次独立的RNA提取、cDNA合成及芯片杂交实验。使用GCOS 1.2软件（Affymetrix）将表达芯片数据标准化，并缩放至信号强度为100。随后采用GeneSpring软件（Silicon Genetics，加州红木城）与Matlab 2009软件（马萨诸塞州MathWorks公司）对基因表达水平进行分析。为鉴定差异表达转录本，使用GCOS 1.2软件（Affymetrix）开展两两比较分析，设置77%的表达变化一致性阈值（即9次比较中至少7次呈现表达上调或下调）；仅满足两两比较检验且表达变化幅度达1.5倍的基因，方可纳入后续分析。