I'm looking to scale up my siRNA reverse transfections from a 6-well to 384-well plate for adherent SH-SY5Y. Should I be scaling the siRNA concentration by surface area in the well, or by the total volume of media in the well?
For background, I have settled on 42pmol in a 6well with 2ml of media on a 9.5cm^2 surface area. That's a siRNA concentration of 21nM. this gives me consistently 50% KO (+/-) 3% SE for all of my targets. It suits our purposes very well, and follows a predictable dose response curve, so i know that the effect is real and that my siRNAs are targeting properly.
When I scale to 384-well, the total volume in each well will be 50ul for on a surface area of 0.056cm^2. So do I scale down my siRNA concentration by nM concentration or surface area?
Lipofectamine / RNAiMAX method appears to work by surface area:
By surface area = 42pmol / (9.5cm^2 / .056cm^2) = approximately 0.25pmol per 384 well
I'm concerned because I'm working with the Viromer Green transfection reagent, and their protocol scales off of nM concentrations. But if I follow this reasoning and suspend 0.25pmol of siRNA in 50ul of media I get a siRNA concentration of 5nM. If I scale off of nM like Viromer suggests, then I would need to plate:
21nM in 50ul media = 42pmol / (2000ul / 50ul) = 1.05pmol per 384-well.
I just think that 1pmol per 384-well seems really high. Am I wrong to be concerned? What would you suggest?
Usually siRNA transfection is reported by concentration, aka people tend to report pmolar/nmolar based on medium amount they use. Considering that in scale up you usually have less volume of media per cell though I'm not convinced that is a good approach, you might end up with significantly higher concentrations per cell (and thus more side effects).
So I'm using per cell ratios for scale UPs (!) and so far it works well. For scale down you might end up with too little per cell (!) if you go by concentration based on media because you tend to have more media per cell in smaller volume. It might depend on your cells though and how well they transfect, how much they are affected by higher cell number in less media (accumulation of metabolites) etc.
So I would suggest you start with per-cell-concentrations and if possible adjust the media to have not too much changes in ratio there. What is crucial though is that you keep the ratio between transfection agent and siRNA the same. Hope that helps.
I always use siRNA concentration no matter how confluence the cell culture is. It is difficult to adjust siRNA based on number of cells on surface and also it is not easy to know how much "effective siRNAs" a cell can take at different concentrations. To make things easier, just stick to the concentration such as 100-200 nM in the medium.
Thank you, Yunfu. It looks like I should just test all three options with a flouro-labeled siRNA, and see which produces the most comparable result.
Dear Martin, when optimizing siRNA transfection protocol (forward transfection) for my screening I started with 12-well and 24-well plates and then went down to 96-well format. Once I set the ratio of cell number to lipid reagent/siRNA complex / well (area) in 24-well plate I simply scale it down to 96-well by using less volumes of plating media, lipid and less pmol of siRNA, but keeping the same final nM concnetration of siRNA/well and the same cell number /area. I scale it down by area, not by nM. You problably have to test how your cells behave/ grow in smaller plate formats first.
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