A Study On Validation Of Cerebral Ischemia-Related Genes Expression Derived From Mouse Exon Array By RT-PCR

Stroke as one of the major diseases which threat to human health is a high prevalence, high morbidity, high mortality rate, high disability of the "four high" disease, which cause great human suffering and bring a heavy burden on families and society. Currently, the pathogenesis research on the cerebral ischemia/hypoxia injury focused on the following aspects, theory of excitatory amino acids toxicity, calcium overload theory, radical theory, and theory of apoptosis, energy metabolism defect and the inflammatory theory. Alternative splicing which widespread in the human genome is a main resource of protein diversity and complexity of gene expression. Research has found that the vast majority of hypoxia signaling pathway genes suffers alternative splicing. However, alternative splicing as a transcriptional regulation of gene expression means, the role and functions in the cerebral ischemia/hypoxia injury are still poorly understood.To explore the splicing pattern changes of cerebral ischemia/hypoxia-related genes, and then clearly reveal the regulatory function of alternative splicing, in this study, we first prepared successfully C57BL/6J mice middle cerebral artery occlusion model to simulate the clinical cerebral ischemia. Then, dissection the left and right mouse cerebral cortex and hippocampus sent to the company with exon array experiments, detected differential expression genes and alternative splicing exons occur at different time points(ischemia 2 hrs, 2 hrs of ischemia/reperfusion 6 hrs), different brain tissues(left and right cortex, left and right hippocampus). Finally, use RT-PCR technology, by designing the alternative exon-specific upstream and downstream primers, to further confirm splicing pattern of cerebral ischemia/hypoxia-related genes. Ultimately, we successfully proved the 14 genes patterns of alternative splicing. Through the 14 gene functional analysis showed that these genes are involved in a series of important physiological processes such as protein translation initiation, protein ubiquitinational degradation, cytoskeleton and mitochondrial energy cycle.To explore large-scale identification of alternative splicing patterns of genes by multiple PCR, and thus accelerates the discovery of a number of gene splicing variants. In this study, we try quintuple PCR with multiple PCR primer design system. First, we selected the 5 reference genes universal expression in mouse brain, using multiplex PCR primer design system design 5 primers, and optimize reaction conditions. In the end, we successfully achieved once PCR on the five target gene identification, and multiple PCR technology laid a basis for the subsequent large-scale identification of splicing patterns of genes.In summary, the preliminary findings of this study revealed alternative splicing plays an important regulatory role in the brain ischemia/hypoxia injury. This study supply important data to understand the brain ischemia/hypoxia injury mechanism and helpful understand the pathogenesis of cerebrovascular disease from the gene alternative splicing level.