Your colleagues submitted interesting questions during the webinar, “High-content genome-wide RNAi screens identify regulators of parkin upstream of mitophagy”; so we’ve collected and answered them here.
Find the answer quickly by clicking your desired question.
- What is the main difference between miRNA and siRNA?
- How should we normalize our siRNA experiments? Should it be based on a positive control such as GAPDH or a scrambled negative control siRNA?
- How many negative controls do you use in your siRNA experiments?
- How can I confirm that the effect of my siRNA of interest is actually knockdown? How can this be distinguished from miRNA effects?
- How can we interpret our results if there is no phenotype observed during an siRNA experiment but a change in total protein amounts as measured between cells treated with siRNAs and those untreated? What can be a possible path forward?
Question 1: What is the main difference between miRNA and siRNA?
Answer: The process of RNA interference (RNAi) can be moderated by either siRNA or miRNA, but there are subtle differences between the two. Both are processed inside the cell by the enzyme Dicer and incorporated into a complex called RISC. siRNA, however, is exogenous double-stranded RNA that is taken up by cells or enters via vectors such as viruses, while miRNA is single stranded and comes from endogenous (made inside the cell) non-coding RNA found within the introns of larger RNA molecules.
Another difference is that, in animals, siRNA typically binds perfectly to its mRNA target, a perfect match to the sequence, whereas miRNA can inhibit translation of many different mRNA sequences because its pairing is imperfect.
Review Carthew (2009) for additional mechanistic insights.
Question 2: How should we normalize our siRNA experiments? Should it be based on a positive control such as GAPDH or a scrambled negative control siRNA?
Answer: We recommend RNAi experiments with a minimum of three types of controls: positive control, negative control, and untreated control. The positive control (e.g., GAPDH gene) monitors the efficiency of siRNA delivery into cells. The negative controls distinguish sequence-specific silencing from non-specific effects. Untreated samples determine the baseline level of cell viability, phenotype, and target gene level.
For normalization of siRNA experiments, we recommend using a negative control. The use of a negative control has an advantage over an untreated control in that it is exposed to the transfection reagent, which factors in an important variable for baselining. We offer array-validated Silencer Select Negative Control No. 1 siRNA for use in siRNA experiments.
Question 3: How many negative controls do you use in your siRNA experiments?
Answer: Our scientists recommend a minimum of one negative control for RNAi experiments. However in large screens multiple negative controls are typically being used by researchers, especially in 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 for the downstream analysis for lead generation.
Question 4: How can I confirm that the effect of my siRNA of interest is actually knockdown? How can this be distinguished from miRNA effects?
Answer: siRNA exerts its effects at the mRNA level, it is more direct to measure knockdown of the target mRNA. We recommend evaluating mRNA knockdown using qRT-PCT based TaqMan Gene Expression Assays. Direct evidence for the siRNA mechanism of action is also established by using a modified 5’-RACE (rapid amplification of cDNA ends) PCR technique, which provides positive identification of the specific mRNA cleavage product (see Soutschek (2004)). We recommend the Invitrogen™ 5’RACE System for this application. This is the most conclusive evidence for a cleavage event guided by a siRNA and is used as a standard in clinical trials.
Question 5: How can we interpret our results if there is no phenotype observed during an siRNA experiment but a change in total protein amounts as measured between cells treated with siRNAs and those untreated? What can be a possible path forward?
Answer: If a phenotype is not observed with the change in protein content, then it is likely that the protein is not involved in the associated morphological change. However if the protein change has been documented with the morphology change then the following factors can contribute to the lack of observance of a phenotype.
- Normally phenotypes are observed if the protein change in a knockdown reagent–treated cell crosses a threshold as compared to the untreated cell. This threshold for the same gene could vary in different cell types. Non-observance of a phenotype suggests that the protein change might not have crossed the desired threshold.
- Morphological changes can be absent if the knockdown reagent is targeting an off-target gene. This lack of specificity impacts other genes in the pathway, which negate the desired effect.
Using a more potent and specific knockdown reagent such as Silencer Select siRNA can lead to higher degree of observable phenotype. We also recommend using positive controls for specific morphological changes (such as siRNAs targeting Kif11 or Plk1 for apoptotic phenotype) as another route to help ensure the integrity of reagents (such a antibodies) being used for monitoring phenotype.