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Define Trasnsfection efficiency interms of siRNA experiment - Sirna uptake Vs transfection efficiency (Feb/16/2009 )

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jiro_killua on Feb 17 2009, 03:44 PM said:

rajgene on Feb 17 2009, 08:02 AM said:

well, interesting points highlighted by all, but all these years i have been educated to use GFP expression as a transfection control(for a plasmid DNA) which directly indicates a functional expression of your transfection experiment.
Then why use this % uptake to relate to transfection efficiency for sirna. its highly misleading for young scientists, who go to cloud number 9 when they see 95% uptake and expect 95% knockdown.
neways thank you all for the input...will discuss about this my next lab meet.


Because the condition of transfecting vector and transfecting siRNA are different....

you are assuming the condition that gives you 95% transfection efficiency in GFP (which is a DNA vector like pEGFPN1) will also give you 95% transfection efficiency in siRNA (which are double stranded RNA)

I'd like to see if you have evidence supporting this assumption in all cells


but before that you have accept the fact that just because 95% of cells have labelled sirnas or with your sirna of ur interest doesn't mean that they are functional in the cell. they have to undergo the endogeneous process of the RNAi pathway which is under cellular control. i have tried it on primary B-CLL cells and cell lines. usually i have an uptake of >70% after 48h but the gfp positive cells is only 30% which directly correlates to my m-rna and protein knock down data!

just thought of an analogy(non-scientific)... imagine that your make cookies/cake in a bakery its looks tasty and jaw dropping, but your boss wont be impressed untill it goes on sale and makes some profit, and that's the efficiency i am talking about.

-rajgene-

To me, using labeled siRNA to study the uptake of cationic polymer or lipid-based transfection agents is a waste of time and $$.

For other methods it maybe different, because it can tell you the entrapment rate of siRNA in the vector, facilitate the separation, or indicate the degree of siRNA protection, etc.

-genehunter-

genehunter on Feb 18 2009, 11:50 AM said:

To me, using labeled siRNA to study the uptake of cationic polymer or lipid-based transfection agents is a waste of time and $$.

For other methods it maybe different, because it can tell you the entrapment rate of siRNA in the vector, facilitate the separation, or indicate the degree of siRNA protection, etc.

cannot agree more gene hunter!

-rajgene-
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