Your colleagues submitted interesting questions during the webinar, “Fundamentals of RNAi Tools and Delivery Technologies”; so we’ve collected and answered them here.
Find the answer quickly by clicking on your desired question.
Q: What is the difference between miRNA and siRNA?
A: The process of RNA interference (RNAi) can be moderated by either small or short interfering RNA (siRNA) or microRNA (miRNA), but there are differences between the two. siRNA is exogenous to mammalian cells, it’s a double-stranded RNA that can be taken up by cells or enters via vectors such as viruses. miRNA are endogenous (naturally present inside the cell) non-coding RNA found within the introns of larger RNA molecules, that go through several steps of processing to produce mature miRNA. siRNA typically has a perfect match to its mRNA target, and induces a cleavage in the middle of a well-characterized complex—leading to reduction of mRNA levels, or knockdown. miRNA is typically fine tuning translation (protein levels) via imperfect base pairing with multiple mRNA targets; their affect on the mRNA levels is more subtle than siRNA and affects many mRNA.
For additional mechanistic insights, see a comprehensive review by Carthew and Sontheimer (2009).
Q: How should we normalize our siRNA experiments?
A: We recommend RNAi experiments include a minimum of three types of controls: positive control, negative control, and untreated control.
- The positive control typically targets a constitutively expressed housekeeping gene like GAPDH and serves to monitor the efficiency of siRNA delivery into cells.
- The negative controls are designed to be non-targeting with little to no sequence similarity to known genes and serve to distinguish non-specific effects from sequence-specific silencing activity.
- Untreated controls determine the baseline level of cell viability, phenotype, and target gene level.
For normalization of siRNA experiments, we strongly recommend using a negative siRNA control. The use of a negative control has an advantage over an untreated control in that it is exposes cells to the siRNA complexed with the transfection reagent, which factors in an important variable for establishing a baseline effect. To this end, we offer the Silencer™ Select Negative Control siRNAs that are designed to have no significant sequence similarity to mouse, rat or human genes and that have been assessed by microarray analysis to demonstrate minimal effects on gene expression.
Q: How many negative controls do you use in your siRNA experiments
A: Our scientists recommend a minimum of one negative control for RNAi experiments. However in large screens multiple negative controls are typically used by researchers, especially in cell-based assays that show high variability. Use of multiple negative controls in high-throughput screens allows for understanding the variability and noise in the biological assay and so that it may be taken into consideration in the downstream analysis for lead generation.
- Carthew RW, Sontheimer EJ. (2009) Origins and Mechanisms of miRNAs and siRNAs. Cell 136(4):642–655.
- Soutschek, J. et al. Therapeutic silencing of an endogenous gene by systemic administration of modified siRNAs. Nature 432, 173-178 (11 November 2004) | doi:10.1038/nature03121
- Soutschek J et al. (2004) Therapeutic silencing of an endogenous gene by systemic administration of mocified siRNAs. Nature 432:173–178.
FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES.
For Research Use Only. Not for use in diagnostic procedures.