摘要
目的通过实时定量PCR(real-ti me PCR)检测基因表达的mRNA,建立一种直接观察小干扰RNA(si RNA)抑制目的基因表达的方法。方法化学设计合成对应于FTH1(ferritin heavy chain1,FTH1)基因表达mRNA的si RNA,经TurboFectTMin vitro Transfection Reagent转染小鼠RAW264.7巨噬细胞,48h后,提取总RNA,逆转录为cDNA。以3-磷酸甘油醛脱氢酶(glyceraldehyde-3-phosphate dehydrogenase,GAPDH)为内参,定量检测FTH1的表达,比较干扰前后FTH1基因mRNA的量。结果 3种si RNA处理的小鼠巨噬细胞中FTH1基因的表达抑制率最高为97.60%。结论实时定量PCR方法的建立为研究布鲁氏菌在侵染巨噬细胞过程中FTH1的功能提供了有效途径。
The aim of the present study was to establish an assay of real-time PCR to detect the inhibitory effect of siRNA on gene expression.The chemically synthesized siRNAs matched with ferritin heavy chain 1（FTH1）mRNA were transfected into RAW264.7 macrophages with TurboFectTM in vitro Transfection Reagent.Then the total RNA was extracted from the cells and reversely transcribed into cDNA.The expression of FTH1 was quantified and compared by real-time PCR on the basis of expressions of g1yceraJdehyde-3-phosphate dehydrogenase（GAPDH）as internal control in different groups.Three kinds of siRNAs could significantly inhibited FTH1 mRNA expression in mice RAW264.7 macrophages and the inhibition rate reached to 97.60%.Results demonstrated that the built real-time PCR could be employed in detection of FTH1,which provides a method to study FTH1 function in the course of Brucella infection.
出处
《中国人兽共患病学报》
CAS
CSCD
北大核心
2010年第7期638-641,共4页
Chinese Journal of Zoonoses
基金
国家自然科学基金(30800813)
兵团博士基金(2009JC15)
高层次人才科研启动资金专项(RCZX200828)
973项目(2010CB530200)联合资助
