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236 Cheng and Chang3.4. Functional Assessment of shRNA-Expression Vectorsin Mammalian CellsTremendous evidence has already shown that not all of the RNAi-targetingsequences selected from a particular gene exhibit the same potencies on inducinggene silencing. Only a limited number of trigger siRNAs are capable ofinducing highly efficient target gene silencing in a sequence-specific manner.The silencing efficacy of siRNAs is dependent on the specificity of the targetsites within the gene and can only be determined experimentally based on theinhibition of the target-gene expression. Several widely used approaches can beused to analyze the efficiency of gene silencing induced by DNA vector-basedshRNA expression, including (1) Northern blot, (2) quantitative reverse transcription(RT)-PCR, (3) Western blot, (4) immunostaining, and (5) functionalactivity assay (see Fig. 6). In general, the effect of gene silencing can bedetected 24–48 h after transfection, dependent on the abundance and the stabilityof the proteins encoded by the target genes.3.4.1. Transfection of shRNA-Expression Vectors1. Subculture and plate 1 × 10 5 cells per well in 2 mL growth medium onto a six-wellculture plate 24 h before transfection. For immunostaining, cells are plated on aglass cover slip in 2 mL growth medium in a six-well culture plate 24 h beforetransfection.2. Transfect 2 µg of shRNA-expression vector, or cotransfect 0.5 µg of RNAi-targetgene-expression vector and 1.5 µg of trigger shRNA-expression vector by usingLipofectamine 2000 following the manufacturer’s protocol.3. Incubate the transfected cells at 37°C in a CO 2incubator for 48 h.3.4.2. Isolation of Total RNAs for Northern Blot or RT-PCR1. Remove growth medium and wash the transfected cells three times with PBS.2. Harvest the transfected cells from the plate by using cell scrapers or spatulas intoa 50-mL culture tube.3. Purify total RNAs from the transfected cells by using TRI reagent following themanufacturer’s protocol.4. Perform Northern blot or RT-PCR analysis with specific probe or primer pairaccording to standard protocols, respectively.3.4.3. Preparation of Total Cell Lysates for Western Blot1. Remove growth medium and wash the transfected cells three times with PBS.2. Harvest the transfected cells from the plate by using cell scrapers or spatulas intoa 50-mL culture tube.3. Prepare total cell lysates from the transfected cells by using protein lysis buffercontaining protease inhibitors.4. Perform Western blot analysis with specific antibody according to standard protocols.