Gene expression profiles of E13 bladder neck/urethral compartments (GUDMAP Series ID: 25)

The long term objective is to create an encyclopedia of the expression levels of all genes in multiple components of the developing bladder. The central thesis is straightforward. The combination of micro dissected tissues and FACS sorted cells plus microarray analysis offers a powerful, efficient and effective method for the creation of a global gene expression atlas of the developing urogenital system. Microarrays with essentially complete genome coverage can be used to quantitate expression levels of every gene. The ensuing rapid read-out provides an expression atlas that is more sensitive, more economical and more complete than would be possible by in situ hybridizations alone. The data submitted here delineates the gene expression profiles of the mesenchymal and epithelial compartments of the E13 mouse bladder neck/urethral compartment. FVB/N mice were time mated. At embryonic day 13 mice were euthanized by decapitation and the bladders were microdissected and cut just below the ureters. Since the EDTA dissociation was ineffective, the bladder neck and urethra were collected and treated with 1 mg/ml trypsin in Tyrode's solution for 20 minutes at 37°C. The layers were separated using a fine needle and rimming. The samples were placed in RLT and stored at -80°C. RNA was prepared and the Epicentre 2-round amplification scheme described under the Lessard Group Protocols on the GUDMAP pages were performed. The amplified RNA was examined with the Affymetrix GeneChip Mouse Genome 430 2.0 Array.本研究的长期目标为构建发育中膀胱多组成部分的全基因表达全景图谱。本研究的核心论点简明清晰：将显微切割组织、荧光激活细胞分选（Fluorescence Activated Cell Sorting, FACS）所得细胞与基因芯片（microarray）分析相结合，可为发育泌尿生殖系统构建全局基因表达图谱提供一种高效、可靠且极具优势的方法。具备全基因组覆盖度的基因芯片可定量检测所有基因的表达水平，其快速的信号读出结果可获得比单纯原位杂交（in situ hybridization）更灵敏、更经济且更完整的表达图谱。本次提交的数据刻画了胚胎期13天（E13）小鼠膀胱颈/尿道区的间质与上皮组分的基因表达谱。实验采用FVB/N品系小鼠进行定时交配，于胚胎第13天通过断头法实施安乐死，显微切割获取膀胱并于输尿管下方截断。由于乙二胺四乙酸（Ethylenediaminetetraacetic acid, EDTA）解离效果不佳，收集膀胱颈与尿道组织，于37℃下在台氏液（Tyrode's solution）中以1 mg/ml胰蛋白酶处理20分钟。通过细针分离与边缘修剪实现组织分层，将样本置于RLT缓冲液中并保存于-80℃。随后提取总RNA，并按照GUDMAP网页中莱萨德课题组实验方案所述的Epicentre公司两轮RNA扩增流程进行扩增。对扩增后的RNA采用Affymetrix GeneChip小鼠基因组430 2.0芯片进行基因表达检测。