12 replies to this topic

[rajgene]

Hi All,
i have a problem. we routinely use sirnas to knockdown genes of interests in our lab. we also the use the fluorescently labelled sirna to check the sirna uptake.
recently my lab mate and me had an argument regarding the definition of 'transfection effciency' during an sirna experiment.

Her stance was that she got -95% transfection efficiency- upon facs analysis using fluorescently labelled control sirna, my stance was that she had only -95% uptake- of sirnas(which is very good) but cannot say it as a transfection efficiency, since 95% uptake doesn't reflect 95% knock down.

in my own experiments i have achieved over 75% sirna uptake, but only 30% knockdown of target gene or 30% expression of pGFP(as a control)
my questions are
1. what is the best way to measure the tranfection efficiency for an sirna experiment.
2. do you agree with my stance or my labmate' stance

thx for your time.
Raj

P.S definition efficiency of any system e=(output/input)

[genehunter]

You are correct. Uptake which can be achieved rather easily, does not equal to the rate of transfection, because siRNA in the complex taken up by cells needs to be freed to be active and quite large % of siRNA in the complex was not freed in fact.

[genehunter]

Assay for specific gene knock down is the only real measurement.

[it_never_ends]

If you look at the definition of transfection, it goes: -
"The process by which exogenous DNA in solution is introduced into cells. OR. The introduction of foreign DNA into eukaryotic or prokaryotic cells".

So, I would say transfection efficiency is the % of cells that the exogenous siRNA has been successfully introduced into. It doesnt translate into "Knockdown efficiency" which is the % of reduction in mRNA/protein of the targeted gene.

[genehunter]

You have some points at the surface, but you forgot to consider the fact that in real world transfection is a process of delivery of "functional" nucleic acid into cells.  Getting DNA to cells to near 100% efficiency is not that hard, but that never translates into transfection efficiency.  For DNA, transfection rate is % cells express the reporter gene. and I think it is reasonable to extend to siRNA, according to its biologic activity, which is gene kockdown.

Most of DNA/siRNA delivered (or taken up by) cells were inactivated, or compartmentalized and non-functional.

Edited by genehunter, 16 February 2009 - 10:54 AM.

[jiro_killua]

genehunter, on Feb 16 2009, 11:49 AM, said:

You have some points at the surface, but you forgot to consider the fact that in real world transfection is a process of delivery of "functional" nucleic acid into cells.  Getting DNA to cells to near 100% efficiency is not that hard, but that never translates into transfection efficiency.  For DNA, transfection rate is % cells express the reporter gene. and I think it is reasonable to extend to siRNA, according to its biologic activity, which is gene kockdown.

Most of DNA/siRNA delivered (or taken up by) cells were inactivated, or compartmentalized and non-functional.

% Uptake > % Transfected > % Knockdown

% here refers to % of cells

Flow Cytometry measures % Uptake (cells containing the fluorescent siRNA), while Realtime PCR measures % knockdown, but we don't know about % transfected because we cannot assume the efficiency of your siRNA to be 100%

Only if your siRNA can 100% knockdown your gene when successfully transfected, we can say % transfected = % knockdown

[genehunter]

I agree with you on that in principle. However, there is no meaningful ways to tell which is transfected siRNA, which is not , other than see through an indirect measure by its activity, which is not 100%.  I wonder if gene silencing for any stable transfected cell lines can get near 100%,  at least for some targets.

[jiro_killua]

genehunter, on Feb 16 2009, 01:09 PM, said:

I agree with you on that in principle. However, there is no meaningful ways to tell which is transfected siRNA, which is not , other than see through an indirect measure by its activity, which is not 100%.  I wonder if gene silencing for any stable transfected cell lines can get near 100%,  at least for some targets.

Totally agree with you

What I mentioned is just for theoritical purpose

Personally, I only care about % intake and % knockdown

And the only reason I care about % intake is, when I see no knockdown, I want to check whether the siRNA was being intake by cells at all

% Knockdown is the ONLY BIOLOGICALLY meaningful observation, and % intake is only TECHNICALLY meaningful

Edited by jiro_killua, 16 February 2009 - 03:16 PM.

[rajgene]

jiro_killua, on Feb 16 2009, 04:15 PM, said:

genehunter, on Feb 16 2009, 01:09 PM, said:

I agree with you on that in principle. However, there is no meaningful ways to tell which is transfected siRNA, which is not , other than see through an indirect measure by its activity, which is not 100%.  I wonder if gene silencing for any stable transfected cell lines can get near 100%,  at least for some targets.

Totally agree with you

What I mentioned is just for theoritical purpose

Personally, I only care about % intake and % knockdown

And the only reason I care about % intake is, when I see no knockdown, I want to check whether the siRNA was being intake by cells at all

% Knockdown is the ONLY BIOLOGICALLY meaningful observation, and % intake is only TECHNICALLY meaningful

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.

[jiro_killua]

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

Edited by jiro_killua, 17 February 2009 - 04:26 PM.

[rajgene]

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.

[genehunter]

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.

[rajgene]

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!