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:

  1. 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
  2. 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.
    • Untransfected 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 
  3. 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.

  4. 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.
siRNA - Resources
  • Find product guides, FAQs and more.
Video: Choosing a Dharmacon siRNA product line and format
  • Sometimes having too many choices is more difficult than having too few. We are here to help.