Gene regulation by Sox9 in the conjunctival epithelium

Wild type or Sox9 fx/fx; Le-Cre-positive (Sox9-/-) conjunctival epithelium from the temporal, nasal and dorsal region of E14.5 mouse embryos was laser micodissected from three embryos of each genotype. Total RNA was purified and reverse transcribed and amplified using a NuGEN kit. cDNA was biotinylated and hybridized to Illumina MouseWG-6 v2.0 bead arrays. Three wild type and three Sox9 knockout embryos were used. cDNA obtained from each region of the conjunctival epithelium was pooled and used for microarray analysis. Statistical analysis was performed using the values from the 25-50 beads on each array.我们从两种基因型（野生型与Sox9 fx/fx; Le-Cre阳性，即Sox9-/-基因敲除型）的各3枚胚胎发育第14.5天（E14.5）小鼠胚胎的颞侧、鼻侧及背侧区域的结膜上皮组织中，通过激光显微切割（laser microdissection）分离得到目标样本。随后对总RNA（total RNA）进行纯化，并使用NuGEN试剂盒完成逆转录（reverse transcription）与扩增。将所得互补DNA（cDNA）进行生物素标记后，与Illumina MouseWG-6 v2.0微珠芯片阵列进行杂交。本实验共使用3枚野生型胚胎与3枚Sox9基因敲除胚胎。将来自结膜上皮各区域的cDNA混合后，用于芯片微阵列分析。统计分析基于每张芯片阵列上25至50个微珠的信号值完成。