Analyzing the Response of RNAi-Treated Drosophila Cells to Death Stimuli by Quantitative Real-Time Polymerase Chain...

A useful complement to animal studies is the use of Drosophila cell lines to analyze cell-death responses. There are numerous Drosophila cell lines available, such as S2 cells, which possess the advantages of being semi-adherent, fast growing, relatively robust, and useful for transfection and knockdown studies, whereas other lines, such as mbn2, are more suitable for analyzing hormone-induced cell death and gene expression. Drosophila cell lines are very amenable to knockdown studies as the cells take up double-stranded RNA (dsRNA) from the medium, initiating gene silencing and resulting in a high level of gene knockdown. This means that the cell lines are useful for investigating the response to death stimuli, following gene knockdown, by examining the expression of cell-death genes. This protocol describes the synthesis of dsRNA for treatment of Drosophila cells and the subsequent analysis of cell-death gene expression by quantitative real-time polymerase chain reaction (qPCR).