I want down regulate the expression of my gene using specific siRNA, purchase from Dharmacon. I tried to use different siRNA concentration to optimize my protocol of transfection (I use an electroporation method, following anufactor instruction) on my model system (embryonic neural progenitor cells), but in any case I obtain a good reduction of my gene of interest (about 60-70%), but the same situation it's observe also in the presence of scramble siRNA! I don't observe a reduction of mRNA expression, but a reduction of protein, also in western blot than in immunofluorescence
Maybe check if this happens in another cell line; and then check that your manufacturer hasn't sent two batches of your targeted siRNA. You could also test another scrambled control in your cells.
You could try to use a transfection reagent to get the siRNA into the cell. The electroporation could be causing the knockdown of your protein.
You don't necessarily need to use the DharmaFECT reagent suggested in the Dharmacon protocol. You can try it with any transfection reagents that you may have in your lab. (Preferably an siRNA specific reagent).
Remember....
The mRNA and protein levels fluctuate with differently after the transfection of siRNA. mRNA knockdown will occur at 24-48hrs. mRNA levels may begin to return to normal after this time.
Protein knockdown will occur at 48-96 hours due to the siRNAs not targeting proteins directly. Make sure you analyse the mRNA and protein levels at the appropriate times.
You may need to do a time course to analyse the best termination times for mRNA and protein.
thanks a lot for your answers!
I tried to do a immunofluorescence analysis in a time course from 48 to 96h post-transfection, and observe a protein level reduction at 48h. My problem it's the scramble! So now I contact the costumer service of Dharmacon and attend their answer
As Yvonne suggested perhaps the transfection process is causing this reaction. Transfection can be stressful for the cell and each cell type may react differently. Compare the levels between
1) Scrambled RNAi cells with DharmaFECT,
2) Mock treated cells- Only contains the DHarmaFECT no scrambled sequence,
3) Non-treated cells- no DharmaFECT no scrambled sequence.
Using this combination you may be able to understand what is affecting your cells.
Hello
I agree with yvonne try a "mock" transfection
http://jbx.sagepub.com/content/early/2015/04/04/1087057115579638.full.pdf 115523.html
http://www.biotechniques.com/protocols/Electroporation/Transfection-of-siRNA-or-Plasmid-DNA-Into-Primary-Cells-Using-the-Gene-Pulser-MXcell-Electroporation-System/biotechniques-
http://www.sabiosciences.com/newsletter/rnai.html
Hi
Some times some of the scramble siRNA may have off target, so you can try other scramble RNA from dharmacon. I recommend you to use any other siRNA transfection reagent (like lipofectamine or dharmafect) instead of Electroporation and include mock transfection (without any siRNA just transfection reagent). May be these things will give answer whether transfection itself causing a reduction in target protein or non specific target of scramble siRNA.
Good luck.
I have had significant knock down (80-90%)when I used Dharmacon transfection reagents and 20 NM siRNA on macrophages. Toxicity was minimal.
I agree with Yvonne that you tyr a mock transfection. However, I feel that for gene silencing it may be preferable to use transfection methods which are minimally stressful on the cells compared to electroporation which can be stressful and cause off-target effects. I would also suggest doing a time course for optimal gene silencing effects
The Targefect siRNA kit works very effiiciently for transfecting siRNA (transfection efficiency usually >90%) even into very difficult to transfect cell types, also works well for co-delivery of siRNA and plasmid DNA and has been used in several publications (selected list of citations and product link is given below
https://targetingsystems.net/pdf/siRNA.pdf
https://targetingsystems.net/citations.php#
If you would like to efficiently deliver (functional or fluorescent) siRNA in vitro (cell lines) or In vivo (animal models), an advanced technology is based on pH-responsive, multifunctional hybrid nanoparticles of carbonate apatite. For more information, you may visit the company (NanoFlex LLC) website (https://nanoflex-usa.com).
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