Top three tips for troubleshooting your RNAi experiment

Troubleshoot your RNAi experiment in three easy steps

Are you new to RNAi experiments and need some guidance on troubleshooting?

Our team, consisting of PhD trained scientists with over 50 years of combined technical support experience, has put together their top three tips for getting your RNAi experiments working successfully.

These suggestions are presented in the order in which you would encounter them during an experiment, not necessarily their order of importance.

By taking these three factors into consideration, your RNAi experiments should perform as expected. If not, please contact technical support.

shRNA siRNA
Step 1:
Initial Experimental Setup

Have the experimental conditions for shRNA lentiviral particles been optimized?

  • Try decreasing serum in the transduction media and include transduction enhancers like polybrene.

Has functional titering been performed?

  • Every cell line is different, and titer needs to be calculated in the cell line in which the experiments are to be performed.

Did resuspension of the siRNA pellet go as planned?

  • By following this protocol, you will ensure complete resuspension of your siRNA pellet. If your siRNA stock concentration is not correct, the minimum amount of siRNA required for silencing may not be delivered to your cells. If there are any issues with this step, contact technical support for further guidance.
Step 2:
Delivery

Are the cells displaying the expected antibiotic resistance and reporter gene expression?

  • Optimization of delivery may be required.

How is the functional positive control performing?

  • Knockdown of the target of the functional positive control at the mRNA level, as assessed by qPCR, demonstrates that the shRNA system is functional in the cell line of interest.

Have a range of Multiplicities of Infection (MOIs) been tested?

  • Different genes and cell lines will require different expression levels of the shRNA. A higher MOI may be required to see the knockdown needed.

How is the functional positive control performing?

  • It should give >80% knockdown at the mRNA level when assessed by qPCR while maintaining >80% viability. The percent knockdown of the positive control is equivalent to the transfection efficiency, telling you if your siRNA is being efficiently delivered. If the transfection efficiency is less than 80%, further optimization of the delivery conditions may be needed.
Step 3:
Detection

How is knockdown being measured?

  • RNAi acts by causing the degradation of mRNA, which is directly assessed by qPCR, so this is the most quantitative, reproducible, and applicable way to assess siRNA or shRNA performance. Assays like Western blotting and FACS measure downstream effects, which can be greatly influenced by factors like antibody specificity and rates of protein turnover. For this reason, we always recommend initially assessing siRNA and shRNA performance by qPCR. Once you know your siRNA is working well, then move on to the protein level or phenotypic assessment of your choice.

Additional Resources