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What to do Post-transfection of cells with siRNA?


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#1 cm13

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Posted 21 November 2011 - 08:12 AM

I am currently attempting to optimise transfection of siRNA into endothelial cells.
I could maybe use some help.

My cells are at 70% confluency and then trypsinised.
I'm using a microporator with different siRNA samples, so the cells are gathered and used with a range of the following siRNA volumes:

10, 20, 30, 50, 100nM and a scrambled siRNA control in a six well plate.
Each sample uses 500000 cells per reaction.



Now, once transfected, here's where i'm not sure what to do.
Do i need to change the media a certain number of hours after transfection?

Also, how long after transfection should i wait to investigate knockdown?
Six, twenty-four, forty eight?

I'm aware that after a certain time, the cell gets rid of the siRNA and the protein of interest gets synthesised again.
But i'm not too sure on how long to wait.

I plan on RNA extraction,which i'll then make cDNA out of, and do real-time PCR on the samples. Is that the best way of investigating the knockdown (lysis of cells would mean i'm doing western blotting on too many blots)

Has anyone any advice?

#2 tea-test

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Posted 21 November 2011 - 09:36 AM

in my opinion, if you do not encounter high toxicity on the cells you don't have to change the medium at all.

After 24h I observed a good silencing effect which was also maintained after 72h, but if you are not sure you can do a time course, for instance, 6h, 24h, 48h, 72h.

real-time PCR is a good way to look for the silencing effect. If you want to confirm by western blot be aware that there is a delay in the knock-down on the protein level compared to mRNA.
tea-test: The artist formerly known as Ned Land

#3 cm13

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Posted 21 November 2011 - 09:43 AM

How long of a delay?




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