Can Free Flow Electrophoresis Improve RNA Separation with No Gel or Dyes?

[DAN1010]

I am looking for some advice on the experiment described below.

I am attempting to use electric potential to move negatively charged RNA toward a positive electrode.

The procedure is similar to traditional electrophoresis except there is no gel or dies. I attempt to describe the setup below.
A transparent 8mm diameter by 20 cm long transparent PVC pipe with a L shaped attachment on either end. Filled with about 15 mL of pH 8 buffer cocktail. Platinum wires are used as electrodes at either end of the tube and the DC power supply ranges from 25-200 Volts.
Procedure: I pipette in .07 mg of trulia yeast RNA next to the negative electrode and apply a voltage. After some time, I pipette out 1 mL of fluid (buffer solution with some RNA) from the positive electrode and look for a 260 nm peak in a UV spectrometer. For a control, I repeat the experiment however do not apply a voltage.

I have tried the experiment at voltages ranging from 25 - 200 Volts (200 volts melted my tube)
I have varied the waiting time from 5 minutes to 30 minutes.
I have varied the buffer concentration

Thus far I have not yet seen substantially more RNA at the positive electrode then I get in the control. My original theory was the voltage should push almost all the RNA to the positive electrode but that does not seem to be happening. I have noticed that the absorbance peak of the RNA changes substantially when it is sampled near the positive electrode. The peak drops by as much as 15 nm and the shape changes.

Any suggestions?

 

[Ygggdrasil]

It sounds like you're trying to do capilary electrophoresis. An important consideration here is that the electrophoretic forces must be stronger than diffusion of the RNA molecules in your solution. Electrophoretic forces increase as the diameter of the capilary decreases, and a 8 mm diameter tube may be too wide, especially in a solution instead of a gel (though I don't have direct experience with this technique). Physical chemistry textbooks with a biological bent should have a discussion of the theoretical basis for modeling diffusion versus electrophoresis.

 

[DAN1010]

It sounds like you're trying to do capilary electrophoresis. An important consideration here is that the electrophoretic forces must be stronger than diffusion of the RNA molecules in your solution. Electrophoretic forces increase as the diameter of the capilary decreases, and a 8 mm diameter tube may be too wide, especially in a solution instead of a gel (though I don't have direct experience with this technique). Physical chemistry textbooks with a biological bent should have a discussion of the theoretical basis for modeling diffusion versus electrophoresis.

Thanks for your response.

I have looked into capillary electrophoresis. From what I have seen, the power supplies for capillary eletrophoresis range from 5-30 kV and use capillary tubes with diameters ranging from .025 - .075 mm. I was hoping that having a tube substantially larger, I could get away with using a relatively low voltage power supply.

I think I have have been thinking about my problem wrong, instead of focusing on electrophloresis to move the RNA, I should be focusing on ways to mitigate the diffusion of RNA. That way the control value is small so I will be better able to see the electrophloresis effects.

Are there methods for mitigating diffusion through a tube?

 

[Ygggdrasil]

Are there methods for mitigating diffusion through a tube?

Increasing the viscosity of the buffer might help, but I'm not sure it would lower the diffusion coefficient of the molecules enough to work for your conditions.