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Subsequent DNA and siRNA transfection

plasmid DNA DNA siRNA transfection transient

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#1 xanthippi

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Posted 09 April 2014 - 04:42 AM

Hello all,

 

I need to first transfect MCF-7 cells with plasmid DNA and subsequently divide the transfected population in two and perform two separate siRNA transfections. Does anyone have exprerience with this? I am afraid my siRNA transfection efficiency will be low. Can you think of any additional pitfalls or solutions?

 

Cheers,

Xanthippi



#2 bob1

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Posted 09 April 2014 - 01:15 PM

You could make a stable cell line with the DNA you are transfecting in, then work on the siRNAs separately.



#3 pcrman

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Posted 09 April 2014 - 02:53 PM

We have done transfection with plasmid and siRNA. We transfect them either sequentially (first plasmid and then siRNA as you planned) or in combination. Transfection efficiency is not a big issue.  



#4 xanthippi

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Posted 09 April 2014 - 11:59 PM

hello both, thank you for the answers.

 

Bob1, unfortunately this is not an option. I already have shRNA stables but this particular KD affects cell proliferation and the population that did grow out is very different from the parental cells in unwanted ways!!!!

 

pcrman, thank you for the encouraging words. What siRNA transfection reagents do you use?



#5 bob1

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Posted 13 April 2014 - 12:35 PM

So you are doing a shRNA KD via plasmid and then KD by siRNA as well?



#6 xanthippi

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Posted 15 April 2014 - 06:49 AM

Erm... no! I did the shRNA to simplify things... it didn't work... I went back to the siRNA :) 



#7 bob1

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Posted 15 April 2014 - 12:49 PM

OK - in that case making a stable line and doing the siRNAs after this would work just fine.  Making the line takes a fair bit of time though.



#8 pcrman

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Posted 15 April 2014 - 01:48 PM

We mostly use invitrogen's RNAiMax which has low toxicity.



#9 bob1

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Posted 15 April 2014 - 03:04 PM

Seconded about the RNAiMax - it is excellent.







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