利用RNA干扰技术沉默基因表达在本科实验教学中的设计与实践

Laboratory design and practice for undergraduates: Using RNAi to modulate gene expression

RNA干扰是由双链小RNA介导的基因沉默现象,已成为一个被广泛应用的反向遗传学研究技术。为了让学生更好地理解该技术,本实验教学让学生自己选择靶基因,设计小干扰RNA和引物,然后检测小干扰RNA介导的基因沉默效果。以2018年第五组为例,该组挑选了小鼠长链脂酰辅酶A合成酶1(acyl-CoAsynthetase long-chain family member 1,Acsl1)为靶基因,设计了两对特异性靶向Acsl1 mRNA的小干扰RNA,通过电穿孔的方式将其转染到3T3-L1中,然后提取细胞总RNA和合成cDNA,最后用相对定量PCR检测mRNA的表达量。结果显示两对小干扰RNA都有60%以上的沉默效果。近3年内,大约83%的学生都能独立完成所有实验并最终成功筛选到至少一对有效的小干扰RNA。该教学实践增强了学生对RNA干扰原理和实验的理解,锻炼了学生的实验与科研能力。

RNA interference is a gene silencing phenomenon mediated by short double-stranded RNAs,which has become a widely used research technology for reverse genetics.In order to make students understand the technology better,the students were required to select target genes,to design small interfering RNAs(siRNAs)and primers,and then to test the effect of gene silencing mediated by siRNAs.Taking the fifth group in 2018 as an example,Mus musculus acyl-CoA synthetase long-chain family member 1(Acsl1)was selected as the target gene,two pairs of siRNAs targeting Acsl1 mRNA were designed and transfected into 3T3-L1 by electroporation,then the total RNAs were extracted and synthesized to cDNA,and the expression levels of mRNAs were finally tested by relative quantitative PCR.The results showed that both pairs of siRNAs had more than 60%silencing effects.In the past three years,about 83%of the students completed all the experiments successfully and screened out at least a pair of effective siRNA.This teaching practice for undergraduates enhances students'understanding of RNA interference principle and technology,and exercises students’lab experience and scientific research ability.