RNAi Mammalian Cells Protocols

Cloning of Amplified miRNA Target Sites into 3'-UTR of Luciferase Reporter Vector Protocol Protocol for cloning of amplified miRNA target sites into 3'-UTR of luciferase reporter vector.

Cotransfection of Luciferase Reporter Plasmids with siRNA Duplexes Protocol This reporter assay, for RNAi in mammalian cells, can be used to establish whether the cells under study are susceptible to RNAi.

Detection of RNAi-Induced Protein Knockdown in Mammalian Cells by Western Blotting Protocol Protocol describes the use of western blotting to analyze the RNAi-induced depletion of target protein from mammalian cells.  Immunofluorescence can be used as an alternative.

Immunofluorescence Detection of RNAi-Induced Protein Knockdown in Mammalian Cells Protocol Protocol describes the use of a specific antibody that recognizes the targeted gene product to detect RNAi-induced gene knockdown in mammalian cells.  Western blot technology can be used as an alternative.

Mammalian Cell Culture and Preparation of Cells for RNAi in 24-Well Plates Protocol Protocol introduces a method for maintaining mammalian cell culture suitable for treatment with siRNA.  Transfection of cultured cells with siRNAs and downstream analysis of the knockdown cells.

Oligofection of Small Interfering RNAs in Senescent Human Fibroblasts Protocol Protocol for oligofection of small interfering RNAs in senescent human fibroblasts. This protocol allows the transfection of human senescent cells (regardless of the cause of cellular senescence) with an efficiency of around 70%.

Selection of siRNA Sequences for Mammalian RNAi Protocol To target a specific mRNA for degradation, a portion of the mRNA target sequence must be known, and a segment of the target mRNA must be chosen that will be used for targeting by the cognate siRNA duplex. Protocol describes the selection of siRNA sequences for mammalian RNAi.

Transfection of Mammalian Cells with siRNA Duplexes Protocol Protocol describes a method for the delivery of siRNAs into mammalian cells in the absence of reporter plasmids.  This is best achieved with transfection reagents developed for the delivery of antisense oligodeoxynucleotides.  The quantities of reagents given below are calculated for the transfection of one well of a 24-well plate.