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Wound Scratch Assay using siRNA

siRNA wound scratch assay

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4 replies to this topic

#1 Jester4023

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Posted 06 December 2012 - 04:18 AM

I've been trying to get wound scratch assays working in cells transfected with siRNA but am having problems getting the cells confluent enough. I'm doing reverse transfections using RNAiMAX as my cells are difficult to transfect and normal forward transfection doesn't seem to cut it.

I've done plating efficiency to figure out how many cells I need to plate in order to get a confluent monolayer to scratch, but the cells don't seem to spread out after transfection. They'll adhere, but remain quite rounded. I've tried different cell concentrations, but still the same problem. There are plenty of cells there, but they're all rounded up and not filling in the gaps on the plate.

I've tried setting up the transfections in larger plates and trypsinizing and replating them into the scratch plates when you would normally put post-transfection media on. I've tried putting the larger plates into fewer, smaller wells for the scratch but that's not working either. I finally got what looked like enough cells and a nice monolayer today, scratched the plates and went to image them and all the cells had washed off the plates.

I can't really leave the plates much longer before I scratch because I run the risk of missing the knockdown window from the siRNA.

Any suggestion at all would be greatly appreciated!
Thanks
Jess

#2 bob1

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Posted 06 December 2012 - 12:29 PM

It sounds like the siRNA is having quite a large effect on the cells themselves, which is probably the problem you are having. I don't see any way around this, unless you have assayed the siRNA effect and seen how long it lasts and have a window where the cells will be attached but still show strong KD.

I have used siRNA that have lasted well out to 5+ days post transfection.

#3 pcrman

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Posted 06 December 2012 - 10:53 PM

I agree with Bob on that the siRNA is doing something to your cells. You did not mention whether this thing only occurred to gene specific siRNA transfected cells or also to control siRNA. What concentration of siRNA do you transfect?

#4 Jester4023

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Posted 07 December 2012 - 06:46 AM

Thanks for your replies. It's in the Mock control as well, but if I try to decrease the amount of transfection reagent, I decrease my transfection efficiency (obviously). I'm getting to the point where I'm thinking this is just one of those things. I won't be able to answer the question in this way so I need to come up with something else.

#5 pcrman

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Posted 07 December 2012 - 08:58 AM

In that case, I believe the problem is RNAiMax which generally has low toxicity. You can cut the recommended dose by half. We did a test and found half is just fine in terms of transfection efficiency





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