Dear all,
I wish to find the optimal siRNA tranfection properties for my cell line. I have a fluorescently labelled siRNA which I plan to transfect by using different agents and quantify by using FACS (with iQue instrument). Could anyone share protocols or have any tricks for this? I am new to the field of molecular biology.
Thanks in advance
If your siRNA itself is conjugated with the fluorescent tag i.e. FITC then you can do Side scatter/FITC flow but the result would only signify that siRNA is present in a certain percentage of the cells which may or may not lead to successful silencing. Normally you would do western blotting to see the decrease or complete absence of the protein of interest. Doing flow in this case would not contribute much as long as you have western data.
I agree with Anton Lennikov, having done a similar experiment myself. I didn't use FACS but I did live cell confocal fluorescent imaging to make sure the siRNA is inside the cells, captured multiple images to ascertain % of transfected cells, then did a qRT for the expression of gene of interest, if mRNA was low as I expected I would confirm with a western to determine effect time, i.e. how long does it take for decreased mRNA expression to reflect on the protein level which usually depends on the protein half life.
If you are new to flow cytometry analysis; these links may help you get started and are relevant to your experimental design:
https://www.flowjo.com/learn/flowjo-university/flowjo/before-flowjo/2
https://pubmed.ncbi.nlm.nih.gov/14976370/
https://www.biolegend.com/en-us/live-dead
https://www.bdbiosciences.com/en-us/applications/research-applications/multicolor-flow-cytometry
(I would also recommend including a viability marker when analyzing your samples so you can distinguish between all 4 populations: live+transfected, live+untransfected, dead+transfected, dead+untransfected).
Hi,
The objective of using flow cytometry is to quantify the amount of si rna taken up by the cells. And I guess u want to silence a protein of your interest.
In that case I will suggest you to do a dual staining one with your si rna flurosence and the other with the antibody of the protein of your interest. if you have two different flurophores such as Fitc for sirna and secondary antibody with Tritc conjugated. The dual staining will give you an idea about the percentage of cells taking up si rna is getting silenced or not.
The other is to do a western as suggested above
All the best
If you're interested in general optimization, FACS using a dye-labeled oligo is an option. Be careful though since your oligo may stick to the outside of your cells, so make sure your cells are well washed prior to fixation. However, I think your best approach is to use a validated siRNA and confirm its efficacy on its cognate transcript by RT-qPCR as mentionned above. Alternatively, WB as an indirect proxy of mRNA abundance could be used, but it has limitations (protein half-life, sensitiviy/abundance issues etc..).
BOC Sciences's siRNA transfection reagents are efficient, low toxicity, ideal siRNA transfection system for mammalian cells.
https://transfection.bocsci.com/products/rnai-transfection-kits-3757.html
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