Hello, I am having trouble to find shRNA or siRNA to knockdown ATG5 or ATG12 protein (human). Tried many commercial ones, but still did not find anyone that works.
Anyone who has successfully knockout down these two proteins? what kind of methods you use? any suggestions?
Thanks,
This is more molecular biology, and not really bioinformatics.
How about searching through publications to see if others have done so successfully (and if so, which shRNAs/siRNAs/protocols they used).
It might not be a problem with your shRNAs/siRNAs. Many cells are extremely difficult to transfect and you really need to optimize your transfection conditions.
It depends a lot on how you're doing the transfection and what cell lines you're using. Try different methods of transfection: Lipofectamine, calcium phosphate, polyethylenimine, etc. Try forward transfection vs. reverse transfection. Try transfecting multiple times. Try serum-reduced media w/ no antibiotic. Try titrating the transfection reagent (and amount of siRNA/shRNA). Use a positive control for transfection efficiency of your cells -- e.g. a fluorescently tagged control siRNA or an siRNA that you know works. Use different assays to measure whether your knockdown is successful -- qPCR, western blot, or change in cell viability/proliferation relative to your negative control cells.
Maybe try doing a stable shRNA expression (e.g. lentiviral).
Optimizing takes a lot of trial and error. If you're just going about throwing siRNAs onto cells and hoping to see a phenotype, you're not likely to be successful.
In addition to trying to optimize the experimental set-up, check databases of RNAi screens like Mitocheck or GenomeRNAi for observed knock-down phenotypes.
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In my experience, cell viability and proliferation are too unspecific phenotypes to be useful. There really is only one way of knowing if the knock-down is successful and that is measuring the residual protein level. If the effect is strong, a carefully done Western blot may be enough to convince you or you can monitor reduction in a GFP-tagged version of your target (ideally from a tagged genomic locus because overexpression may be hard to knock-down)