快速筛选芳香烃受体基因特异RNA干扰序列的初步研究

目的筛选能诱导人芳香烃受体(AhR)基因表达沉默的特异RNA干扰片段。方法根据RNA干扰设计的原则,选择4个片段,用聚合酶链反应(PCR)方法,以含有人U6启动子的质粒为模板,分别扩增正义和反义小干扰RNA(siRNA)表达盒,纯化后直接转染人支气管上皮细胞(16HBE),抽提细胞总RNA,定量竞争逆转录PCR(RT-PCR)检测目的基因的mRNA表达差异,确定RNA干扰的效率。结果含AhR的干扰序列a493、a671、a1337和a1885的siRNA表达盒对AhR的mRNA表达抑制率分别为61.6%、-0.5%、78.6%和95.4%。结论运用PCR扩增siRNA表达盒从多个候选序列中快速筛选出芳香烃受体有效的干扰序列,为进一步的基因功能研究创造了条件。

Rapid screening of specific RNA interfere sequences for aryl hydrocarbon receptor gene

Objective To screen specific interference sequence which can induce gene silencing of human aryl hydrocarbon receptor(AhR).Methods Four different sequences were selected based on the RNA interference design principle.Each sense and anti-sense small interference RNA(siRNA) cassettes were amplified by polymerase chain reaction(PCR) using plasmid contained human U6 promoter as template.Mixed and purified cassettes were transfected into 16HBE.The total RNA were isolated and employed to quantitative competitive PCR to detect the mRNA expression difference of target gene and to determine the efficiency of interference.Results The average mRNA expression inhibition rates of siRNA expression cassettes containing interfere sequence a493,a671,a1337 and a1885 were 61.6%,-0.5%,78.6% and 95.4% respectively.Conclusion The effective interfere sequence for AhR from candidate sequences was screened by siRNA cassettes amplified by PCR,and offered conditions for studying the gene function of AhR.