Whole heart transcriptional response to complete and epiblast-specfic deletion of Zic3 in 15.5 dpc mouse embryos

In order to better understand the molecular basis for the heart defects seen in Zic3 null and epiblast CKO embryos, we investigated whether complete or epiblast-specific deletion of Zic3 would impact later embryonic heart development at the transcriptional level by whole genome expression microarray. The whole heart was carefully dissected out from 15.5 dpc Zic3 +/y, Zic3 flox/y, Zic3 flox/y; Sox2-cre, and Zic3 -/y embryos, total RNAs were extracted and purified using RNeasy Mini Kit (QIAGEN). Spectrophotometry (NanoDrop-1000 Spectrophotometer, Thermo Fisher Scientific) and microfluidic electrophoresis (Experion Automated Electrophoresis System, Bio-Rad Laboratories) were used for RNA quality control. In vitro transcription was performed using Illumina TotalPrep RNA Amplification Kit (Applied Biosystems/Ambion). cRNAs were hybridized onto Illumina MouseWG-6 v2.0 Expression BeadChips (Illumina) per manufacturer's instructions.为了更好地理解Zic3基因敲除（Zic3 null）以及上胚层特异性敲除（epiblast CKO）胚胎中出现心脏缺陷的分子基础，我们通过全基因组表达微阵列技术，探究Zic3完全敲除或上胚层特异性敲除是否会在转录层面影响胚胎后期的心脏发育。我们从胚胎发育第15.5天（dpc）的Zic3 +/y、Zic3 flox/y、Zic3 flox/y; Sox2-cre以及Zic3 -/y胚胎中小心分离完整心脏，使用RNeasy Mini试剂盒（QIAGEN）提取并纯化总RNA；采用分光光度法（NanoDrop-1000分光光度计，赛默飞世尔科技）与微流控电泳法（Experion自动化电泳系统，伯乐实验室）完成RNA质量质控。使用Illumina TotalPrep RNA扩增试剂盒（应用生物系统/安毕恩）开展体外转录实验，并按照制造商说明书将互补RNA（cRNAs）杂交至Illumina MouseWG-6 v2.0表达微珠芯片（Illumina）上。