Because use of siRNA can affect the expression of the some other gene which is not our interest and may modulate to a phenotype.
Greetings Dr Sabbir Khan, eliminating the off-target effect of siRNA is not possible anyway. how ever, if we take model which having a limited physiological complexity like in vitro cells. well in case higher mammalian models, its highly impossible, but may be possible with targetted drug delivery systems to particular targets organs.
Hi, Sabbir. Why don't you try a rescue experiment? You can put back the activity of your gene of interest by ectopically expressing it and investigate whether the original (wild-type) phenotype is restored.
Conduct a rescue experiment but make sure your expression construct does not contain the siRNA targeting region. It is also standard to show that 2 different siRNAs designed against the same target give the same phenotype.
There is no way to eliminate off-target effects completely. There are several sources of them - sequence-specific off-targeting. You need to make sure that there are no perfect matches between positions 2-18 of the guide strand to other targets. You need to make sure that there are no matches to miRNA seed regions (the major source). You need to have a dose response curve and use the minimal concentration of siRNA to achieve maximal knockdown (usually it will plateau at some level. Make sure there is now excessive cytotoxicity and interferon induction (some sequences induce strong response). Several siRNAs against the same target should produce the same phenotype. If you have several siRNAs use a pool of them - it dilutes sequence-specific off-target effects. Use modified siRNA - they have reduced toll-receptor activation and may prevent risk entry of a passenger strand.
There may be off-targeting effects due to transfection protocol, either independent or combined with specific effect of RNAi. Use control siRNAs and try different transfection protocols. Use self-deliverable siRNAs or shRNAs.
There is no way to get rid of non-specific effects completely, they can be only minimized and controlled to the acceptable levels.
- Badges
- Science topic
- Similar topics
- Geoscience
- Asia
- India