Designing RNAi Experiments
1. Maximize the signal-to-noise ratio in your phenotypic assay
- the number of cells evaluated
- the choice of reagents such as antibodies and/or substrate
- the timing of the assay after siRNA delivery or addition of substrate
- the turnover rate of the targeted gene product
- the effectiveness of the siRNA
2. Identify and minimize edge effects of culture plates
- If your throughput requirements allow for fewer samples per plate, consider avoiding all edge wells in multiwell plates. Instead load those wells with culture medium only or with nontransfected (or non-electroporated) cells.
- Position replicates in the plate so that data from them can be used to evaluate position effects. For example, divide the plate into quadrants and include replicate samples or, at a minimum, positive and negative controls, in different quadrants of a single plate. Alternatively, include replicates in different locations across several multiwell plates.
- Use mathematical or statistical methods to minimize edge effects. Strategic placement of well-defined positive and negative controls can potentially be used to spot faulty data or to normalize data based on location within or among plates. Setting separate requirements for retesting or evaluating ring well data might also be implemented.