When you are designing your RNAi experiments there are a number of considerations. In most cases it will be important to show results using either different methods or using the same method with more than one target. Some other considerations include:
Method of siRNA production
Delivery method(s)/cell type to use
Options for delivering siRNA include transfection and viral delivery. Generally the choice depends on whether the cell type is readily transfectable or if a primary or non-dividing cell type is being used. Also, the choice may depend on whether an animal model system is being used and it is important to deliver in vivo . Note that transfecting for an RNAi experiment is somewhat different than doing an expression experiment. Particularly if protein has long half-life, assays may need to be done at 48 or even 72 hours to get results. Typically you will transfect siRNA or d-siRNA at lower cell density. It is highly important that the transfection efficiency be optimized. Even if an siRNA were100% effective (and most are only 70-95% effective), if only 50% of the cells are transfected you will never see better than a 50% knockdown. Find out about optimizing your transfection experiments
Select appropriate controls
The use of appropriate controls for RNAi experiments is critical. See an outline of the recommended controls to do at different experimental stages
Invitrogen has a wide selection of controls for RNAi. Reagents and kits are available for optimizing and monitoring transfection efficiency, positive and negative RNAi experimental controls, assessing activation of stress responses, and easy selection of the best target sites. Find out which controls to use in your experiments
Measure loss of message or protein function
Verification of RNAi effect requires for either or both the protein and transcript levels to be determined. In some cases an antibody may not exist and then it will be important to carefully quantitate the loss of message. Be aware that although message is knocked down, if protein has a long half-life you may not see an effect. The most common way to verify loss of message is to do real time RT-PCR. This is much more sensitive than RT-PCR. More information on real time PCR.
An end goal of an RNAi experiment is to determine if gene knockdown has a functional effect. A visual assay can be done to identify a phenotypic change , such as looking for apoptotic cells. A lternatively a functional cell based assay can be completed to analyze the effects of gene knockdown , such as a kinase or ion channel assay- or design your own cell based assay using the GeneBLAzer reporter.
Level of knockdown desired
Choosing an RNAi Experimental Approach
Easily harness the power of RNAi in your lab. A table that can help you choose the appropriate method and product to use for fast and easy RNAi experimental design.
For Research Use Only. Not for use in diagnostic procedures.