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siRNA and plasmid cotransfection


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#1 FACS_flow

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Posted 16 November 2009 - 02:42 PM

Hi everyone,

I am currently establishing a co-transfection of miRNA inhibitor and plasmid DNA into a colon cancer cell line.
The only thing with which I am currently struggling with is how to set up a proper
pre-test series to figure out the best transfection conditions.
Ultimately I would like to perform my transfection in 6-well plates as I would need
a significant amount of cells to perform additional assay (like migration assay etc.)

Nevertheless I do not want to waste unnecessary material for setting up the assay so
I thought of performing the setup in 96-well plates as this saves me money, reagent and time.

So here is the question that still bothers me about this 96-well setup thing.
How do I scale up the reagent volume from 96 to 6 well then, what parameter to consider?
Just multiply every amount of reagent by 16 or consider the surface area?
I am still wondering if this is the way to go as I think that 96-well plates and
6 well plates are considerably different.
But on the other hand if I have to consider doing replicates for each condition,
I would be using tons of reagent just for setup if I would go with 6 wells for setup...

maybe someone who already did these kind of setup experiments could share how
she/he succeeded.

Thanks in advance!

#2 Dr Teeth

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Posted 17 November 2009 - 09:34 AM

Hi everyone,

I am currently establishing a co-transfection of miRNA inhibitor and plasmid DNA into a colon cancer cell line.
The only thing with which I am currently struggling with is how to set up a proper
pre-test series to figure out the best transfection conditions.
Ultimately I would like to perform my transfection in 6-well plates as I would need
a significant amount of cells to perform additional assay (like migration assay etc.)

Nevertheless I do not want to waste unnecessary material for setting up the assay so
I thought of performing the setup in 96-well plates as this saves me money, reagent and time.

So here is the question that still bothers me about this 96-well setup thing.
How do I scale up the reagent volume from 96 to 6 well then, what parameter to consider?
Just multiply every amount of reagent by 16 or consider the surface area?
I am still wondering if this is the way to go as I think that 96-well plates and
6 well plates are considerably different.
But on the other hand if I have to consider doing replicates for each condition,
I would be using tons of reagent just for setup if I would go with 6 wells for setup...

maybe someone who already did these kind of setup experiments could share how
she/he succeeded.

Thanks in advance!



I would pretest using a flourescent protein plasmid and fluorescent RNA oligo. What you should be wary of is the ability to transfect plasmid and RNA together does not always mimic the ability to transfect either alone with a given transfection reagent approved for co-transfection. For example, I have found using an RFP plasmid and FITC-RNA oligo that plasmid transfection alone is very high using lipofectamine 2000 in my cell type as is RNA delivery alone; however, a combination of plasmid and RNA yields decent RNA delivery but very poor plasmid transfection and/or expression. It seems to be better if the RNA and transfection reagent are complexed separately from the plasmid and transfection reagent and then combined. Other reagents, such as Dharmafect Duo also appeared to work better for this application in my cell type.
As for scaling up, I haven't had any problems scaling from 24 well up to 6 well, or even 100-150 mm plates, provided you use the correct
factor, adjusting volumes appropriately based on surface area of the well. I haven't tried 96 well, but it should scale up fine. Using an adjustment value of 16, however, is not appropriate. Instead, this adjustment must be based on the surface area of the wells. From what I can find, most 96 well dishes have wells with a diameter of 6.4 mm, yielding a surface area of ~32 mm2. Six well dishes have a 35 mm diameter, yielding a surface area of ~962 mm2.
962 mm2/32 mm2 = ~30--a 30 fold, not 16 fold, difference.

Edited by Dr Teeth, 17 November 2009 - 09:37 AM.


Science is simply common sense at its best that is rigidly accurate in observation and merciless to fallacy in logic.
Thomas Henry Huxley




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