应用Super SMART cDNA合成技术扩增哮喘嗜酸性粒细胞总RNA

Super SMART cDNA synthesis technology for amplifying small amount of total RNA of peripheral blood eosinophils from asthma patients

目的应用Super SMART cDNA 合成技术从嗜酸细胞微量总RNA扩增大量双链cDNA。方法利用Percoll密度梯度离心分离哮喘病人血嗜酸性粒细胞,用Trizol试剂盒提取细胞总RNA,利用Super SMART cDNA合成技术合成cDNA第1链及优化扩增第2链。通过梯度电泳鉴定cDNA产品质量,通过对获得的cDNA定量来评估扩增效能。结果从20 ng总RNA成功扩增出双链cDNA 7.155 μg,电泳结果显示cDNA质量及纯度较高。结论Super Smart cDNA 合成技术可实现从微量总RNA高效扩增高质量的cDNA,为进一步从基因水平研究哮喘机制奠定了基础。

Objective To amplify double-strand cDNA from small amount of total RNA of eosinophils from asthma patients by Super SMART cDNA synthesis technique. Methods The eosinophils were purified from the peripheral blood of asthma patients before and after treatment by Percoll gradient centrifugation, from which the total RNA was extracted using TRIzol kit. First-strand cDNA synthesis and double-strand cDNA amplification were performed using Super SMART cDNA synthesis technique. The quality of the obtained cDNA was evaluated by gradient cDNA electrophoresis, and the amplification efficiency determined by cDNA quantification. Result From 20 ng total RNA, 7.155 μg and 6.568 μg of the tester and driver double- strand cDNAs respectively were obtained successfully, and the result of electrophoresis indicated high quality and purity of the cDNA acquired. Conclusion Super SMART cDNA synthesis technique can effectively amplify high-quality double-strand DNA from a very small amount of total RNA, which may facilitate the exploration of the mechanism of asthma from the genetic level.