Jmjd2a +/+ vs -/- Pituitary microarray

To understand the nature of gene expression differences between wildtype and Jmjd2a knockout females in the mouse pituitary - we performed a microarray on RNA isolated from the pituitary tissue in females who had been induced to kickstart their oestrus cycles. All females (3 per group, virgin and sexually mature) were introduced to the dirty bedding of the same stud male to control for pheromone differences. 24 hours later, the animals were sacrificed by cerival dislocation and pitutiary was harvested. RNA was extracted from the tissue and quality was checked using Bioanalyzer. All RNAs possess RIN quality score of 9.5 or above (10 in most cases). From the resulting data 5 downregulated and 3 upregulated genes were validated by qRT-PCR. While there was expected biological variation to certain degree among individuals, there was a clear distinction in sample clustering between control and knockout samples and good correlation among individual sample genotypes. Of the downregulated genes, two were using immunoblotting on protein extracts isolated from a pool of five pitutiaries derived from females of each genotype subjected to the same experimental design. Briefly,as asummary we observe a strong prominent downregulation of immediate early genes. Virgin mature (8 week age) control and knockout Jmjd2a females were oestrus cycle activated by exposure to dirty bedding of the same stud male (Whitten Effect). Then these females were sacrificed exactly 24 hours later by cervical dislocation and pitutiary tissue was dissected and harvested, washed with PBS and collected in individual eppendorf tubes and frozen in dry ice. The samples were subjected to RNA extraction using the commercial Qiagen RNAeasy kit.