Considerations of an RNAi Experiment

There are several considerations when planning an RNAi experiment. It is important to do the appropriate experimental controls to ensure you have publication quality RNAi data. Other considerations may also be important to your experimental approach.

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

Is a good target sequence known? You may want to use one method to generate initial data or test sequences and another method to do more involved studies or validate results. Especially if a good target sequence is unknown, or if you have had difficulty knocking down your gene using an oligonucleotide approach, you may want to start with an easy, effective method such as transfecting a diced pool of siRNA then validate this data using Stealth™ RNAi , siRNA or RNAi vectors.

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.

Functionality

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

Depending on the gene you are studying the level of knockdown may be important. To be considered a functional RNAi experiment, typically a knockdown of greater than 50% must be achieved. Some targets designed by current algorithms, or validated or guaranteed targets, will only knockdown to 70%. This may not be effective level of knockdown to see a phenotype. Delivery is also critical here. Even if a target is 70% effective, if delivery is less than 100% efficient then the gene knockdown will be lower than 70% in terms of actual results. Ideally, a knockdown of >95% would be desired, but not every gene may have an siRNA that can be designed that will knockdown to this extent. If you are using a target that does knockdown to 70% or 80%, there may still be other potential targets that could result in more effective knockdown. Often addition of a diced pool of siRNA will give very effective knockdown and can be used for comparison.