I have used commercially control and target siRNA from SantaCruz to perform transfection in C2C12 and HSMM. I performed western blot and the target protein was not expressed while the controls were, how to assess the transfection efficiency?
You could isolate DNA and RNA and check their levels using qRT-PCR or qualitatively by PCR, to see at which level there is a problem.
You could isolate DNA and RNA and check their levels using qRT-PCR or qualitatively by PCR, to see at which level there is a problem.
If your transfected protein has a tag (myc/his) you could perform IF and stain for the tag. There could be basal levels of fluorescence but the transfected cells should be much brighter
You keep any plasmid containing GFP taq as a control . Measure the GFP dots in after the trasfection in the trasfected cells which will tells the trasfection efficiency. of your transfection reagent.
You could always do a cotransfection with a gfp plasmid as suggested, however our experience is that siRNAs are taken up more effectively than plasmids. A plasmid cotransfection might therefore underestimate the level of transfection. I advice you to purchase a fluorescent tagged scrambled siRNA and do cotransfections with this.
Cotransfecting a GFP plasmid will not yield accurate results because it is a much larger molecule relative to the siRNA you are transfecting into your cells.
IDT sells a TEX615-labeled siRNA control for use in estimating transfection efficiency. Transfect this siRNA using the same protocol you use for your other siRNA then determine the percent of cells transfected by counting the transfected cells using a Fl. microscope relative to to total number of cells. Hint: ImageJ has a cell count function that allows you to click individual cells that makes this process much easier and less prone to error. This is a peer-review accepted method for estimating transfection efficiency.
You can try FISH to detect it in the cells but basically any molecular beacon that binds your RNA should work.
You can co-transfect a BLOCK-it Fluorescent Oligo from Invitrogen with your siRNA as an indicator of transfection efficiency.
Correct me if I am wrong, but I think it is a expected result that you can't see a protein by Western Blot from the protein lysate from your siTARGET treated cells, but you see it in the one from siNON ? So the silencing efficiency will be the % of siTARGET from siNON.
After all, I don't think it is that much important how much of the siRNA has entered the cell (which will be the true "Transfection efficiency", but did it do what it was supposed to do?
Transfection efficiency is important because it helps you determine whether your siRNA experiment did not work because it was not desiged properly or because it did not get delivered to the cells. It is one of the critical optimization experiments required when developing a transfection protocol. Also, if you report a small effect when using siRNA, miRNA mimics, etc the first thing reviewers ask about is "was the transfection efficiency high enough or are the observed effects simply small due to poor delivery."
Brian, I completely agree about the importance of the transfection efficiency, especially on the note about reporting small effects. I might be lucky that the silencing I do always causes more than 90% decrease in mRNA and protein level, so I didn't have to troubleshoot.
However, if the siRNA is not labeled, I think the more straightforward way to check the delivery is about 4-12 hours post-transfection, and after a VERY, VERY, VERY good wash of the cells, to isolate total small RNA fraction and then detect the target siRNA by tailored PCR. The good wash will be essential as siRNA are usually delivered in the range of 10-50nM, which for a typical siRNA with a mW 13400g/mol corresponds to about 3x10^14 copies per mililiter of media!!!
Revathy, have a look at this link - https://tools.lifetechnologies.com/content/sfs/brochures/cms_083672.pdf - this is a short technical note of how to do that with LifeTechnologies reagents.
In that manual LifeTech actually advise that "To ensure removal of the untransfected siRNA, cells were washed, trypsinized, transferred to tubes, and pelleted prior to lysis". Sounds logical.
Thank you for raising that question!
you have to use a flouro-labeled siRNA. otherwise, you have to assume 100% ko efficiency of the siRNA if you are relying on qPCR or western blot data.
you could try cotransfection with a GFP expression vector coupled with a siRNA targeting GFP. though, i would suggest against it because the data is confounded by seeding density, and the effects of short RNAs on the transfection efficiency of the GFP plasmid. for example, in my tests we tried electroporation and found that our siRNA concentration enhanced transfection efficiency of the GFP plasmid = not good.
There are beacon probes you can use to co-transfect, they can label your siRNA post transfection, similar to FISH assays for example.
https://www.idtdna.com/pages/decoded/decoded-articles/your-research/decoded/2012/04/11/towards-real-time-imaging-in-single-living-cells
Here are more approaches in some overview: http://cdn.intechopen.com/pdfs-wm/6918.pdf
You can always qualitatively quantify the protein running a gel and performing a WB.
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