I am doing an EMSA experiment of protein binding to a fluorescent RNA probe. I am using a native polyacrylamide gel. The RNA is double stranded and I use ThermoFischer's protocol of annealing RNA in a thermocycler for a more controlled cooling (going down a degree per minute until it reaches 25 then switching to 4ºC).

Upon adding protein to RNA probe there does seem to be clear binding; however, there are alot of bands below the free-RNA band. Could this be RNA truncations or un-annealed RNA?

I have shown that if I run the gel longer they will of course run off, but Im more just curious on why this was happening and if anyone had any suggestions to prevent it. I also wanted to note that in the no protein lane there seems to be much less lower bands, could the protein be a factor?