Transcription profiling of mouse E8.5 wild-type and 3 E8.5 Tcof1+/- littermate embryos (Treacher Collins Syndrome)

The object of this study was to identify genes transcriptionally upregulated and downregulated in response to Tcof1 haplo-insufficiency during mouse embryogensis. Experiment Overall Design: Total RNA was extracted from 3 E8.5 wild-type and 3 E8.5 Tcof1+/- littermate embryos using the RNeasy Mini Protocol for Isolation of Total RNA from Animal Tissues (Qiagen) according to the manufacturer's protocol. RNA quality and quantity was determined using the Bioanalyser. To generate targets for microarray analysis, total RNA (100 ng) from each wild-type and mutant embryo was amplified using Two-Cycle Target Labeling (Affymetrix) according to the manufacturer's instructions. Biotinylated target cRNAs (20 ug) from the 3 wild-type and 3 Tcof1+/- embryos were hybridised to separate GeneChip® Mouse Genome 430 2.0 arrays (Affymetrix), following standard Affymetrix procedures.本研究旨在鉴定小鼠胚胎发生过程中，响应Tcof1单倍体不足（haplo-insufficiency）时发生转录上调与下调的基因。 实验总体设计：从3枚E8.5期野生型胚胎与3枚同窝出生的E8.5期Tcof1+/-胚胎中提取总RNA，操作严格遵循Qiagen公司的动物组织总RNA分离RNeasy Mini试剂盒操作流程。利用生物分析仪（Bioanalyser）测定RNA的质量与浓度。为制备芯片分析所需的靶标，本实验按照Affymetrix公司的操作指南，采用双循环靶标标记法（Two-Cycle Target Labeling）对每枚野生型与突变型胚胎的100 ng总RNA进行扩增。将3枚野生型胚胎与3枚Tcof1+/-胚胎的20 μg生物素标记靶标cRNA分别与Affymetrix公司的GeneChip®小鼠基因组430 2.0芯片阵列（GeneChip® Mouse Genome 430 2.0 arrays）进行杂交，实验流程严格遵循Affymetrix公司的标准操作步骤。