I wish to effectively knockdown 2 genes. I have two methods in mind: using both siRNAs at once or doing it sequentially; one knockdown after the other. I intend to proceed with flow cytometry, real time pcr and western blot. Any advice or suggestions?
Dear Ubanako,
When I need to knockout two different genes with siRNA, I set up the following conditions
1) Neg Control siRNA - final conc 40 nM
2) siRNA #1 20 nM + Neg Control siRNA 20 nM
3) siRNA #2 20 nM _ Neg Control siRNA 20 nM
4) siRNA #1 20 nM + siRNA #2 20 nM
This way the total amount of siRNA is the same across all four conditions, and you can simultaneously look at the difference between no knockdown, single knockdown of each mRNA, and then both knockdown as well.
You can use the same theory if you wish to pool siRNA against the same target.
Dear Ubanako,
Do these 2 genes transcribe similar mRNAs?? if yes, then design an RNA interference that would target the transcript sequence where they show more homology. If the genes are not similar have you ever thought about a double transformation event? I imagine this is possible in human biology.....
Dear Ubanako,
I suggest that you try both methods and compare the data. Initially, the sequencially method is better in order to understand better the molecular mechanisms (e.g. modulation of signaling pathways and othere effects due to your first knockdown), then try to see what are the effects with the other one before eventually using both siRNAs. Be aware of false negatives and false positives, be aware about sensitivity and specificity of your siRNAs and be aware of the combinatorial effects of your 2 siRNAs. Best wishes, Farid
Hi Aimone, thanks for your response. The mRNAs are different. Secondly, I already have both siRNAs, separately designed to target 3 regions or their respective target mRNAs.
Dear Ubanako,
When I need to knockout two different genes with siRNA, I set up the following conditions
1) Neg Control siRNA - final conc 40 nM
2) siRNA #1 20 nM + Neg Control siRNA 20 nM
3) siRNA #2 20 nM _ Neg Control siRNA 20 nM
4) siRNA #1 20 nM + siRNA #2 20 nM
This way the total amount of siRNA is the same across all four conditions, and you can simultaneously look at the difference between no knockdown, single knockdown of each mRNA, and then both knockdown as well.
You can use the same theory if you wish to pool siRNA against the same target.
Dear Ubanako,
You can certainly deliver two or more siRNA simultaneously. We do so very frequently. in fact, we often use Dharmacon pools of 4 different siRNA targeted to the same transcript to increase knockdown which is a bit different than the application you desire but it proves the technique is vaible. However, we have also used pools of individual siRNA to different genes. Important considerations are that single siRNA targeted to each mRNA can be less efficient than pools but if you have good individual siRNA to each target you can perform nice combinatorial experiments.
Good luck with your experiments.
Regards,
Tom
Dear Ubanako,
I've delivered more siRNAs before using exactly the same protocol suggested by Charles and it normally works very well. We as well use a lot of Dharmacon smart pools, and never had problems from the transfection of multiple siRNAs together.
Best,
Elena
I have down the double knockdown simultaneosly and confirmed it by Western Blot
I am using the Smart pool siRNA from Dharmacon too and I wonder if combining two smart pools to simultaneously knockdown two genes would be okay?
Double knockdown in RNAi faces problems of efficiency and toxicity of dsRNA (if dsRNA concentrations are high). If you can place both of your dsRNA templates on same vector/bacterium hopefully, it will work.
Dear Charles,
May I ask something from the formula that you suggest
1) Neg Control siRNA - final conc 40 nM
2) siRNA #1 20 nM + Neg Control siRNA 20 nM
3) siRNA #2 20 nM _ Neg Control siRNA 20 nM
4) siRNA #1 20 nM + siRNA #2 20 nM
for no3, siRNA #2 20 nM _ Neg Control siRNA 20 nM,, this mean minus negative control?
thanks for help
RNAi can certainly be used for multi-gene knockdown. Read http://blog.sitoolsbiotech.com/2017/08/understanding-gene-networks-with-combinatorial-gene-knockdown/ for more info.
With siPOOLs, gene knockdown is efficient at low nM and risk of siRNA off targets are greatly reduced. Welsbie et al. has used 4 siPOOLs together with effects replicated with CRISPR.
Please contact [email protected] for more info on siPOOLs.
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