I checked my siRNA knock down efficiency using WB, and my protein A was somewhat knocked down. However, when I did time lapse to see its knock down effects on the other protein B-GFP, the cell did not live well. Its shape changed and the protein B-GFP phenotype was somewhat changed. So, my question is how can u confirm that this phenomenon is due to the siRNA knock down vs its bad living condition (but actually A not KD)?
Are there some kind of shRNA vectors that can be labeled?
A Non-targeting siRNA control is your best bet to discriminate actual KD from general effects of transfection. An untreated control is also essential to evaluate general cell culture conditions. If you see unusual toxicity or phenotypic changes with a non-targeting control, chances are that they are due to transfection rather than a target gene silencing event.
Labeling of an shRNA vector (or a vector that drives GFP expression along with the shRNA; e.g. GIPZ shRNA) is only useful to determine if the shRNA is being expressed; it tells you nothing about whether you have knockdown.
U could also optimize the transfection conditions by reducing lipofectamin or olgofectamin (usually toxic for cells). Another option coul be the use of a different transfection agent.
good luck!!
you definetly need scramble siRNA or GAPDH siRNA to control the effect of the transfection and also the off target effects of siRNA
We did not do the same experiments with a scramble siRNA, but instead not transfected cells. I wonder is there serious difference between scramble siRNA and non-transfected cells. My lab partners do not use scramble siRNAs, but I think it's necessary. I think I should also reduce the lipofectamin. Thank you very much for all your answers.
All the statements make sense, of course. However, it my well be, that off target effects of your siRNA cause a strong phenotype and growth defect, unrelated to the Knock Down of Protein A. Industry labs tend to test multiple siRNAs directed against one target, to tell apart off target effects from the desired phenotyp caused by KD of the designated gene.
However, one must be aware, that every singe siRNA will have off target effects and the only way to circumvent it is to use high complex siRNA pools.
We have made very intersting and promising experiments using high complexity siRNA pools and actually show, that off target effects can be overcome with this approach, leading to a cleaner phenotype and reliable results. don´t hesitate to ask if you want more info on this new approach
Four things (two were mentioned partially):
1. You must use a control siRNA, I do NOT recommend scrambled siRNAs though, they too often have off target effects (seed region binding maybe) instead of being inert or are even immuno-stimulatory. Use proven inert siRNAs like GFP22 from Qiagen or literature described siRNAs against luciferase.
2. Test at least 2 siRNAs with the same target mRNA (but different binding sites). Some companies offer validated siRNA sequences, meaning they have been tested for activity. So I would go with this to save time. Once again, Qiagen has some, not sure of other companies (I'm not affiliated with Qiagen in any way).
3. Make sure you don't overload the cells with the siRNA, there are several publications out there showing nicely that too much siRNA perturbates the endogenous microRNA pattern of the cell and such causes all kinds of side effects. 4. If your transfection reagent/condition causes damage to the cells that will throw off your results too. You can always try another transfection reagent (while avoiding re-branded same ones of course). If you use Lipofectamine, try for example TransIT TKO in parallel.
Like any other experiments, positive and negative controls are essential in siRNA works. You may want to try some controls that are offered by different companies (they have mentioned to you Qiagen) and I'm giving you another suggestions that is Dharmacon. Non-sense (non-targeting) siRNA is important, however, try to find something that has been proven in the literature to have a low off-target effects. Additionally, positive control is phenomenal because it gives you confidence in how the efficiency of the depletion was. Once again, in siRNA experiments, many factors can affect the work including: cell seeding density, transfection reagents, lipid concentration, positive and negative controls, transfection period, type of transfection (reverse or normal), and siRNA concentration. If you are sure about all these conditions then your transfection should work fine :).
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