利用RT-PCR技术验证基于小鼠外显子芯片发现的缺血相关基因的表达

目的:利用RT-PCR技术验证并确认基于小鼠外显子芯片发现的部分缺血相关基因的表达,以鉴定候选基因的外显子是否发生可变剪接,从而实现对外显子芯片结果的鉴定。方法:根据生物信息学分析结果,选取小鼠外显子芯片中的3个基因(Ube3c,6330439K17Rik,Atp7a),在预测发生可变剪接的外显子两侧设计上下游引物,PCR后进行凝胶回收,再克隆到载体中进行测序。结果:RT-PCR及测序结果表明,Ube3c基因在6号外显子、6330439K17Rik基因在12号外显子、Atp7a基因在3号外显子发生可变剪接,与芯片预测结果一致。结论:RT-PCR技术可针对外显子芯片的结果进行可靠性验证,为可变剪接基因表达研究提供了一种有效手段。

Study on Validation of Ischemia-Related Genes Expression Derived from Mouse Exon Array by RT-PCR

Objective：To validate and confirm the expression of ischemia-related genes from mouse exon array using RT-PCR technology for identification candidate gene alternative exon.Methods：According to bioinformatics analysis,three genes（Ube3c,6330439K17Rik,Atp7a） in the mouse exon array were selected.The upstream and downstream PCR primers were designed flanking the predicted differently expressed alternative spliced exons.The PCR products were separated by 1.5% agarose gel and cloned into the vector for sequencing.Results：Our data showed that the 6th exon of Ube3c,the 12th exon of 6330439K17Rik and the 3rd exon of Atp7a were alternatively spliced.These results were consistent with exon array.Conclusion：The RT-PCR technique for the exon array reli-ability analysis provides an effective means for the study of the alternative splicing genes expression.