I'm trying to knockdown my protein in adherent cells and label cells with the fucci system for cell-cycle segmentation using microscopy. My protocol is as follows
Day 0 : siRNA reverse-transfection on trypsinised cells
Day 1: siRNA transfection on adherent cells
Day 2 : Collect cells for western/imaging
I was wondering if it's ok to co-transfect/transduce the viral vectors on Day 1, so the cells are labelled when I collect them on Day 2.
It would also be great if someone has tried IF after transfecting with the fucci vectors and if the tags survived the fixation protocol.
Thanks for your help
Your protocol doesn't mention transduction - I'm assuming you're meaning that on Day 1 you're transducing the cells. In general, if you're not worried about recording the transfection efficiency after each step, then you should be able to do co-transfection with lipid-based reagents (https://altogen.com/product/altofect-transfection-reagent/). You'll probably have discrepancies in transfection efficiency, but it shouldn't result in many issues, and the tags should show up the next day.
Hi Nisha,
As you know, often times it is very difficult to achieve high knockdown efficiency when using an viral delivery approach. May I ask if you have tried Self-Deliverable RNA silencing FANA Molecules to achieve high knockdown efficiency?
If not, I encourage you to review the attached FANA delivery protocol without the use of any transfection reagents.
Please kindly let me know if this can help your specific experiments?
Have a good day!
Your protocol doesn't mention transduction - I'm assuming you're meaning that on Day 1 you're transducing the cells. In general, if you're not worried about recording the transfection efficiency after each step, then you should be able to do co-transfection with lipid-based reagents (https://altogen.com/product/altofect-transfection-reagent/). You'll probably have discrepancies in transfection efficiency, but it shouldn't result in many issues, and the tags should show up the next day.
Hi Nisha,
The cells don't like to be transduced/transfected two successive day, the efficency of the 2nd time generally drastically drops. So my advice is to do co transfection/transduction, (or wait 4-5 days after the first transfection/transduction but in your case I think it is difficult to do that)
Regards,
Nicolas
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