A brief guide of points to consider when planning an siRNA experiment
Within the last 20 years, small interfering RNA (siRNA) has become a routine tool in many molecular biology labs for performing gene knockdown experiments to study gene function. Some straightforward experimental practices have emerged to avoid common pitfalls. Take a look at these four main points to consider for successful siRNA experiments:
- Choose appropriate siRNA reagents and delivery methods that are adapted to your cell type
- Are your cells easy to transfect? Or are they difficult, such as primary or suspension cells? Conventional siRNA reagents can be delivered with standard lipid-based transfection reagents, or used with an instrument-based delivery method (e.g. electroporation) if cells are difficult to transfect.
- A novel option for difficult-to-transfect cells is Dharmacon Accell siRNA, which is chemically modified for passive self-delivery into cells that are resistant to transfection reagents, such as primary neurons, immune-derived cells, and in vivo systems
- Use appropriate positive and negative controls in each and every experiment
- Controls are essential to optimize and monitor transfection and assay conditions, and distinguish non-specific effects. Each experiment should include the following minimal controls in triplicate.
- A positive control to monitor transfection success in each experiment. It is essential for effective target knockdown to have a good transfection efficiency, as it is the most variable factor in siRNA experiments. Data should be consistent between replica. Generally, we see 95% or higher knockdown levels with our validated positive controls under optimized conditions. Efficiency below 80% indicates further optimization is needed.
- A non-targeting negative control siRNA to distinguish sequence-specific silencing from non-specific effects. It is also used to normalize your gene expression data.
- Un-transfected cells to obtain information about the effects of siRNA transfection on gene expression, phenotype and cell viability.
- Check out our application note to learn more: Effective controls for RNAi experiments
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Optimize siRNA delivery conditions
- It is often underestimated, but you may be required to screen a few conditions to achieve robust assay results.
- In case you are performing lipid-based transfection, our siRNA transfection protocol includes valuable guidelines for optimization, such as testing several cell densities and amounts of lipid reagent. We provide DharmaFECT Transfection Reagents 1-4 for lipid-based transfection of siRNA and small RNAs. These four specific formulations that are optimized for efficient delivery into various cell types.
- Finally, you will choose conditions that are reproducible and show a high siRNA delivery level in your cell type (as measured by your positive control siRNA) and low level of cell toxicity.
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Confirm your knockdown on the mRNA level using qPCR
- While it is good experimental practice to analyze your knockdown with multiple assays, and on multiple levels, always make sure to confirm knockdown of your target at mRNA level--even if it is not your desired final readout. The reason is simple: siRNA targets mRNA for degradation. Therefore, qPCR is the most direct detection method to measure siRNA-mediated target knockdown. qPCR has another big advantage: It is quantitative! This makes troubleshooting and optimizing experimental conditions much less complex than protein-based data. This is why we base our functional guarantee on mRNA data. For qPCR pointers and troubleshooting please see our article on qPCR basics.
- Once you detect a reasonable knockdown of your mRNA, you can further optimize time courses to follow up on protein production or phenotypic changes. In case your follow-up experiments include western blotting, please consider that protein stabilities are highly variable. A normalization control and standard curve will be required for accurate knockdown estimation.
Additional resources
- RNAi Controls
Critical to any gene silencing or microRNA modulation experiment, RNAi controls enable accurate interpretation for reliable, reproducible results. - siRNA - Applications
Understanding siRNA and how to use it for RNAi - siRNA - Products
Dharmacon siRNA products are the result of scientific innovation in siRNA design and novel modification strategies to optimize potency, specificity and delivery.