Dear colleagues, how many scrambled siRNAs do you use in case when you either target single gene with two or more siRNAs or target multiple genes with multiple siRNAs?
Especially if you are building a phenotypic assay, I would look at several (4-5) and pick the one that is the most reliable under your condition (closer to the transfection reagent alone). Then, targeting your gene of interest by 2-3 non-overlapping siRNAs sequence will boost your confidence that your effect is likely coming from its down-regulation (You should validate at the protein or mRNA level)
Dear Dr. Brosseau, thank you for the reply. I'm about to perform a gene expression dependence test, not a phenotypic assay. But yes, I will use 2 non-overlapping siRNAs to target the gene. Hence, the most confusing question now is - should I use 2 respective scrambled siRNAs, or using only one of them is just enough?
Well in our case, we use a universal siRNA (same length, same chemistry) that we use in every experiment what ever the gene we target. It is not a "scrambled" sequence. In any case, you have to BLAST the control to make sure that as a minimum of theoric target on your transcriptome of interest
For the transient knockdowns you are performing, use a control siRNA that targets a gene that doesn't exist in the genome of the species you are working with. For example if knocking down genes in mammalian cells i use siRNA-GL2 that targets the luciferase gene which is only present in the insect genome. First, use two siRNAs against your target sequence, and validate by immunoblot or other convenient means. If both perform satisfactorily, the two are enough as a bare minimum, considering that all experiments in science require three independent repeats. Otherwise get some more siRNAs. The more the better. Alternatively, you can pool two or more individual siRNAs together to enhance the phenotypic difference. Good luck
Thank you, JP and David. I think I should use maximally non-targeting siRNA of the same chemistry as target siRNAs.
I agree with David that control siRNA may be better if they are design to target a gene that doesn
I would avoid the use of scrambled control siRNAs as they don't generally constitute an effective control. Scrambling the sequence of your targeting siRNA may unintentionally yield a sequence that is highly effective at knocking down other gene(s). Your control siRNA should be of the same chemistry, capable of entering RISC (i.e. behaves like a normal siRNA), and non-targeting. Control siRNAs designed against genes that don't exist in your species of interest may have unintended off-target effects on genes that do exist in your species of interest. Thus, the ideal control siRNA for your proposed studies is one that is designed not to have off-target effects on any genes in your species of interest, or at least to target as few as possible. Most companies refer to these as non-targeting control siRNAs, and they are usually designed using a combination of in silico and genome-wide expression approaches.
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