国家自然科学基金资助项目31172164。
目的 筛选用于比格犬ERβ基因RNA干扰实验的细胞和检测所用引物。方法 根据比格犬ERβ氨基末端区域和DNA结合区设计三对引物，用阳性质粒筛选最佳引物应用于RNA干扰效果检测，并利用293T、Hela和vero细胞的cDNA验证该引物的特异性。对这三种细胞内人ERβ基因的存在情况也进行了检测。结果 经过三次重复性实验之后，结果显示引物C36684扩增效率高，且没有非特异条带，该引物对293T、Hela和vero细胞cDNA扩增时同样没有非特异性条带，但293T细胞中存在人的ERβ基因。结论 引物C36684用于检测RNA干扰效率，而Hela细胞则作为RNA干扰实验的细胞工具。
Objective To screen the cells and construct primers to be used for RNA interference experiment of ERβ gene in Beagles.Methods Three pairs of primers were designed according to the amino terminal and DNA binding area of ERβ gene in Beagles, and the best primers were screened as the primers to be used to detect RNA interference effect by positive plasmid, and the cDNA from 293T, Hela and Vero cells was used to validate the specificity of the primers. The existence of homo ERβ gene was also tested in the three cell types. Results After experiments repeated for three times, the results showed that they had high amplification effect and there was no nonspecific amplification by the C36684 primers. The amplification results of cDNA from 293T, Hela and Vero cells also showed that there was no nonspecific amplification by the primers, but there was homo ERβ gene expression in 293T cells. Conclusion The C36684 primer pair is selected for detecting RNA interference effect, and Hela cells are selected as the tool for RNA interference experiment.
钟睿,甘艺,赵彦斌,刘冰,孙兆增,朱宇旌,栾新红,胡仲明,张勇,曾林.比格犬雌激素β受体RNA干扰实验细胞及PCR检测引物的筛选[J].中国比较医学杂志,2014,24(2):42~45,51.
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