View - ResearchGate

DNA Vector-Based shRNA-Expression Systems 237Fig. 6. Experimental procedures for assessing the inhibition efficiency of shRNAexpressionconstructs. (A) Seeding of targeting cell line. The targeting cell line is subcultured24 h before transfection and plated into six-well culture plate at 1 × 10 5 cellsper well. (B) Transfection of shRNA-expression construct. The cultured cells are eithertransfected with 2 µg of shRNA-expression construct or cotransfected 0.5 µg of RNAitarget-gene expression construct and 1.5 µg of trigger shRNA-expression construct byusing Lipofectamine 2000 according to the manufacturer’s instructions. (C)Assessment of inhibition efficiency. After 48 h incubation, the transfected cells are harvestedand lysed for either RNA or protein level analysis of target-gene expression byusing Northern blot, RT-PCR, Western blot, immunostaining, or functional reporterassay (luciferase activity).