How can we verify/check up the effectiveness of RNA interference? (using molecular methods)?
Thanks,
The hallmark of RNAi is effective knockdown of the target gene expression.
Let us assume you have a target, TNF, that needs to be suppressed. The most widely used techniques are qRT-PCR, quantitative estimation of the gene products (mRNA and protein).
In some cases, it might be important to monitor the level of protein as well because even though the mRNA level might be low without a concomitant decrease in protein level, it might suggest that the protein turnover is less.
For example, if you have an siRNA directed against TNF, then you essentially check the level of expressed TNF by using a primer specific to this gene product. You can run a positive control in which you treat the cells/animals with TNF-inducing compounds such as LPS and normalize your results.
You might want to include at least three housekeeping genes in your setup for several reasons. Some of the reasons could be that these serve as internal controls to indicate nucleic acid extraction, quality of samples, quality of PCR. Also, when you are using the deltadeltaCt method, you would need these to normalize your data.
While this is just the tip of the iceberg, the list of methods can be a bit more comprehensive.
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology
- Molecular Methods