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miRNA/siRNA transfection doubts


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#1 losybelle

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Posted 25 April 2011 - 08:48 AM

Hello all!

I'm starting a project in which i have to transfect some microRNAs and siRNAs into HELA cells. I'm gonna use Lipofectamine 2000 and 24 well's plates. have a couple of doubts that maybe, you guys, can help me to resolve. The first one is that for transfections in HELA cells when i was using promoter vectors, i used to add around 70.000 cells per well. Is this amount ok for miRNA and siRNA transfections?

The other thing is that i've seen that in lipofectamine 2000 manual there are two different protocols depending if you are using a vector or a siRNA. But i need to use the two of them at the same time for a luciferase assay (vector- reporter + siRNA); do you know if i can use the plasmid lipofectamine protocol for doing this transfection?

Thank you very much!!

#2 pcrman

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Posted 25 April 2011 - 07:01 PM

The same principal regarding cell density for plasmid transfection applies to siRNA/miRNA transfection. A cell density of 60-80% should work depdending on the speed of cell growth and duration of transfection.

You can transfect plasmid and siRNA together or one after the other. For reporter assay, probably it makes sense to transfect the plasmid first, the the siRNA/miRNA.

#3 losybelle

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Posted 26 April 2011 - 08:42 AM

The same principal regarding cell density for plasmid transfection applies to siRNA/miRNA transfection. A cell density of 60-80% should work depdending on the speed of cell growth and duration of transfection.

You can transfect plasmid and siRNA together or one after the other. For reporter assay, probably it makes sense to transfect the plasmid first, the the siRNA/miRNA.


Thank you!! :)




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