摘要
目的建立一种利用实时荧光定量聚合酶链反应(RT-QPCR)技术检测RNA干扰(RNAi)效果的方法 ,为筛选有效的RNAi提供一种科学、可靠的实验方法。方法选用质粒载体pavu6.1-27构建的3个携带NCOA-3基因干扰序列和一个阴性对照序列的重组质粒,转染ECA109细胞,培养48 h,在荧光显微镜下观察荧光细胞的数目,计算转染效率,然后收集细胞提取总RNA,RT-QPCR检测各组转染细胞NCOA-3基因mRNA表达量,从而计算各组的干扰效率。结果相对于A组未转染细胞,各组mRNA表达量分别为B组101.3%,C组85.5%,D组69.8%,E组72.2%,C、D、E组于A组和B组差异均有统计学意义(P<0.05),而A组和B组相比差异无统计学意义(P>0.05),计算得到3个干扰载体的干扰效率分别为39.0%、82.1%、75.6%。结论 RT-QPCR能进行RNAi效果的筛选,其自动化程度和检测灵敏度高;本研究建立的利用RT-QPCR筛选有效RNAi片段的方法可以为肿瘤研究、药物筛选、功能基因组研究等各个领域提供可靠的实验基础。
Objective To establish a method to detect the effect of RNA inte ference on time flurogenic quanli tative polymerase chain reaction（RT-QPCR）and offer a scientific and reliable method for screening out effective se quence for specifical RNA interference. Methods Three recombinant plasmids constructed pavu6.1-27 taking differ ent inteference sequences for NCOA-3 were selected. And a negative control sequence was also inserted into plasmid vector pavu6. 1-27. These plasmids were transfected inlo ECAI09 cells respectively. The numbers of fluorescence cell were overviewed under fluorescence microscope 48 hours later, and the transfecting efficiency were calculated. Then cells were collected to extract the total RNA and the mRNA level of NCOA-3 was detected by RT QPCR,and then the inlerfcring efficiency of each class was calculated. Results Compared with class A,the expression level of NCOA- 3 of each class was B 101.3% ,C 85.5% ,D 69.8% ,and E 72.2% respectively. The difference of C（D,E） and A had a statistical significance（P〈0.05）. But the difference was of no statistically remarkable difference when B compeared with A （P〉0. 05）. The interference efficiency of each class was C 39. 0%,D 82. 1%,and,E 75. 6% respectively. Conclusion On-time flurogenic quantitative polymerase chain ceaction could be used to detect the effect of RNA inteference. The automaticity and sensitivity of this method were significant. This report would offer a scientific and reliable method for the research on tumor,drug screening and functional genome study,and so on.
出处
《检验医学与临床》
CAS
2010年第10期903-905,共3页
Laboratory Medicine and Clinic
