Voltage

Where your thermal cycle times the same as theirs? What about the voltage across the gel and the time of the run? Did you expose the RNA to light too much? It's possible you denatured them. Also, there may have been RNAses in your extract that could have ...

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... as much if you want to destain, which should not even be a problem with gelred. So why not post stain? If this is really not an option, lowering voltage is usually a good start. And re-using buffer less.

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... we used ethidium bromide. I already have some ideas what to try to improve the separation (using TBE buffer, using 0.5x buffer, applying lower voltage, etc.) Since I optimized several methods in the last months, I am really tired of it and I just wanted to ask you if you had the same problem ...

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... it is too thick to fit into a western transfer machine), but I think you would have a very hard time figuring out the right parameters (current, voltage, time) at which to transfer it.

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hmm, just a random thought, but maybe your voltage is too high when you're doing the transfer. the smaller proteins, that move faster, might simply go through the membrane. I have never heard of this happening anywhere though, but then again, ...

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