Gene regulation by Pax6 in the lens placode - E9.5 (3)

Wild type or Pax6 fx/fx; Le-Cre-positive (Pax6-/-) surface ectoderm from E9.5 mouse embryos was laser micodissected from three embryos of each genotype. Total RNA was purified, pooled for each genotype and triplicate samples were reverse transcribed and amplified using a NuGEN kit. cDNA was biotinylated and hybridized to Illumina Mouse6v2.0 bead arrays. Three wild type and three knockout embryos were used. The RNA from embryos of the same genotype was purified and pooled and triplicate samples were amplified and used for microarray analysis.本研究从E9.5期小鼠胚胎的野生型（Wild Type）或Pax6 fx/fx; Le-Cre阳性（Le-Cre-positive，即Pax6基因敲除（Pax6-/-））的表面外胚层（Surface Ectoderm）中，每种基因型取3枚胚胎，通过激光显微切割（Laser Microdissection）获取样本。随后提取总RNA（Total RNA），将同基因型的RNA合并后制备三份重复样本，采用NuGEN试剂盒进行反转录与扩增。将合成的cDNA进行生物素标记，随后与Illumina Mouse6v2.0微珠芯片进行杂交。本实验共使用3枚野生型胚胎与3枚基因敲除胚胎，同基因型胚胎的RNA经提取、合并后，制备三份重复样本进行扩增，并用于芯片微阵列分析。