Smad泛素化调节因子2 siRNA的筛选及转染原代肾小管上皮细胞条件的优化

Abstract

Abstract: To lay the foundation for investigating the role of smurf2 in aristolochic acid nephropathy,we optimized the best transfection condition of Smurf2 siRNA,and screenned the best siRNA fragment,then realized the Smurf2 transient gene silencing. We cultured renal tubular epithelial cells (RTECs),Lipofectamine^TM 2000 was taken for transfection medium,transfecting Smurf 2 siRNA into RTECs,the transfection condition was optimized by observing expressions of green fluorescences and undertaking Real-time PCR reactions.After transferring the Smurf2 siRNA, mRNA inhibition ratios were detected by Real-time PCR.We detected the effects of compound Smurf2 siRNA on RTECs activity by CCK-8;at the same time,we detected the expression of Smurf2 under different sites of Smurf2 siRNA by Real-time PCR and Western blotting. The results showed that the best transfection condition was 1.5 μL Lipofectamine^TM 2000 and 30 pmol siRNA,the transfection efficiency was 70% to 80%.Smurf2-619 siRNA was proved with the most obvious interference effect.Smurf2 protein levels decreased significantly in the group of Smurf2 siRNA compared with the normal group （P<0.05）.We determined the best transfection condition of Smurf2 siRNA, Smurf2-619 siRNA had the highest inhibition rate for the expression of Smurf2.

Key words: Smad ubiquitination regulatory factor 2; transfection; siRNA; renal tubular epithelial cells; optimization

ZHAO Hai-jiao,GAO Lei,LU Yan,TIAN Ya-hui,JIA Zhi-hua,TIAN Hai-yan,WU Guo-juan,ZHANG Zhong-wen. Screening and Transfection Condition Optimization of Smurf2 siRNA in Renal Tubular Epithelial Cells[J]. , 2014, 41(1): 19-24.

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Screening and Transfection Condition Optimization of Smurf2 siRNA in Renal Tubular Epithelial Cells

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